bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Antagonism and Nicotine‐Induced Tolerance/Cross‐Tolerance Among nAChR Agonists in Mice
Authors: de Moura Fernando Barreto , McMahon Lance Richard
Published: November 2023

β2* nAChRs mediate the behavioral effects of nicotine; the extent to which β2* nAChRs also mediate the behavioral effects of other nAChR agonists including FDA approved smoking cessations aids (e.g., varenicline) has not been established. The goal of this study was to identify differences in nAChR mechanism(s) underlying the behavioral effects of nicotine, epibatidine, varenicline, and cytisine in male C57Bl/6J mice (n=7). Mice responded under an FR20 schedule of liquid food delivery; rectal temperature was assessed before and after operant conditioning sessions. The effects of nAChR agonists were tested alone, in combination with mecamylamine or DHβE, and after daily nicotine treatment consisting of 3 doses per day, 1.78 mg/kg per dose, 90 min apart. Nicotine, epibatidine, varenicline, and cytisine dose‐dependently decreased rate of FR responding; the respective ED50 values were 0.44 mg/kg, 0.0027 mg/kg, 2.8 mg/kg, and 4.0 mg/kg. Nicotine, epibatidine, varenicline, and cytisine dose‐dependently decreased rectal temperature; respective decreases in rectal temperature at the largest doses studied were −5.9°C, −3.4°C, −3.5°C, and −3.4°C. Mecamylamine (1 mg/kg) significantly antagonized the rate‐decreasing and hypothermic effects of each agonist. DHβE (3.2 mg/kg) antagonized the rate‐decreasing and hypothermic effects of nicotine and epibatidine, as evidenced by a maximum 3.5‐fold rightward shift in the nicotine and epibatidine dose‐response functions. In contrast, DHβE did not antagonize the effects of varenicline and cytisine. Daily nicotine treatment produced significant rightward shifts in the nicotine dose‐response functions (i.e., tolerance) of 4.1‐fold for rate‐decreasing effects and 4.0‐fold for hypothermic effects, as well as significant rightward shifts in the epibatidine dose‐response functions (i.e., cross‐tolerance) of 2.2‐ and 2.9‐fold, respectively. In contrast, daily nicotine treatment did not significantly modify the potency of varenicline and cytisine to produce either rate‐decreasing or hypothermic effects. To the extent that DHβE is a selective β2* nAChR antagonist, these results suggest that β2* nAChRs mediate the rate‐decreasing and hypothermic effects of nicotine and epibatidine, whereas non‐β2*, mecamylamine‐sensitive nAChRs mediate the effects of varenicline and cytisine. Chronic nicotine treatment appears to decrease the responsiveness of β2* nAChRs, but not the responsiveness of the non‐β2*, mecamylamine‐sensitive nAChRs that mediate the effects of varenicline and cytisine. These results suggest that nAChRs mediating the effects of varenicline and cytisine are different from the nAChRs mediating the effects of nicotine and epibatidine.

Support or Funding Information

Supported by USPHS grant DA25267
Identification of LMAN1 as a Druggable Cellular Target for Reduced Secretion of Matrix Metalloproteinase‐9
Authors: Duellman Tyler , Burnett Jack , Yang Jay
Published: November 2023

Matrix metalloproteinase‐9 (MMP‐9) is a secreted endopeptidase playing a major role in shaping the extracellular environment. Elevated levels of secreted MMP‐9 have been implicated in the pathogenesis of a number of diseases and thus a detailed understanding of the secretory mechanism of this enzyme could help identify novel cellular targets to prevent or reverse disease conditions that result from excess levels of secreted MMP‐9. Previous reports have characterized some of the early secretory events of MMP‐9 showing that this glycoprotein follows the canonical secretory pathway entering the quality control folding cycle in the endoplasmic reticulum (ER) before transport of the properly folded protein to the Golgi apparatus and beyond for secretion. Despite this knowledge there is limited information about the other secretory events including the mechanism by which MMP‐9 exits the ER. CRISPR‐mediated knockout of the ER resident carrier protein LMAN1 (Lectin, Mannose‐Binding 1) confirm its functional role in the efficient secretion of MMP‐9. Through a complementation assay we determined that LMAN1, a well‐studied lectin‐carrier protein, interacts with the secretion‐competent fully N‐glycosylated MMP‐9 in the ER. Our study suggests LMAN1 selectively interacts with the properly folded, fully N‐glycosylated MMP‐9 within the ER and shuttles it to the ER‐Golgi Intermediate Compartment (ERGIC). This study has identified a novel cellular target in LMAN1 that can be therapeutically targeted for inhibition of MMP‐9 secretion. Importantly the MMP‐9: LMAN1 complementation assay provides a valuable tool for the development of a fluorescence‐based high‐throughput drug screening assay aimed at finding small molecules capable of interfering with the LMAN1: MMP‐9 interaction and thus reducing the secretion of MMP‐9.

Support or Funding Information

This study was supported by the Clinical Translational Cardiovascular Training Grant T32 HL07936‐12 (TD), Bamforth Endowment Fund (JY) from the Department of Anesthesiology, UW Madison, and NIH RO1 GM107054 and GM105665 (JY).
Small Molecule Inhibitors of Constitutively Active Gsα Exert Varying Effects on Receptor‐Mediated Signaling
Authors: Rylaarsdam Robin Pals , Carroll Julie , Poch Mark , Schumacher Morgan
Published: November 2023

McCune‐Albright Syndrome is a rare genetic disorder caused by mosaicism for a constitutively active allele of Gsα. Patients exhibit a broad range of hyperendcrinopathies, skin hyperpigmentation, and polyostotic fibrous dysplasia of bone. The bone disease is particularly difficult to manage clinically. We hypothesized that designing a molecule that preferentially binds to the mutated form of Gsα could provide the starting point for drug development and better therapy for the bone disease. Previous work in our lab identified the region near R231 as a potential druggable target site on the mutated version of the protein. In silico docking analysis provided a slate of candidate molecules to test for inhibition of the MAS protein's constitutive activation of adenylyl cyclase. Two molecules, 6‐oxo‐N‐{[2‐(pyrrolidin‐1‐yl)pyridin‐4‐yl]methyl}‐1,6‐dihydropyridazine‐3‐carboxamide and N‐{[3‐(3‐methylphenyl)phenyl]methyl}‐6‐oxo‐1,6‐dihydropyridazine‐3‐carboxamide were able to significantly and in a dose‐dependent manner reduce cAMP levels in HEK cells transiently transfected with the constitutively active Gs‐R201H allele.. Neither of these molecules significantly reduced basal cAMP levels in cells transfected with wild‐type Gs constructs. Cellular responses to receptor‐mediated adenylyl cyclase activity were unaffected by the first molecule, while the second drug reduced the maximum cAMP response to luteinizing hormone receptor activation but did not affect the EC50 of the response.

Support or Funding Information

This work was supported by NIH grant 1R15ED020190‐01 and the Scholl Foundation Endowed Chair in Natural Sciences.
A High Throughput Screening Campaign for Inhibitors of RGS6 and RGS17
Authors: Bodle Christopher , Hayes Michael , Wu Meng , Fisher Rory , Roman David
Published: November 2023

G Protein Coupled Receptors (GPCRs) are highly targeted proteins for drug discovery, with over 50% of all drugs on the market targeting these receptors. Regulator of G Protein Signaling (RGS) Proteins regulate the signaling cascade of GPCRs by accelerating the intrinsic GTPase activity of G protein alpha subunits and returning the heterotrimeric G protein to its inactive state. Recently RGS proteins have been implicated as being important in a variety of disease states. RGS17 has been identified as being inappropriately upregulated in both lung and prostate cancer. Knockdown of RGS17 resulted in decreased cell proliferation in vitro, and reduced tumor mass and volume when compared to control when cell lines were injected subcutaneously in nude mice. RGS6 has been shown to play an essential role in doxorubicin mediated cardio toxicity, has been shown to have an important role in anxiolytic and depression phenotypes in mice, and has been implicated as a critical mediator of behavioral and pathological responses to alcohol. Here we present the screening of two RGS proteins (RGS6 and RGS17) using Alpha Screen technology against the NCI Next Diversity Library. Assay robustness was determined for both proteins, with RGS6 Z‐factors above 0.7 and RGS17 Z‐factors above 0.6. Approximately 20,000 compounds were screened against RGS6 resulting in identification of 20 hit compounds, and roughly 60,000 compounds (including the 20K also screened against RGS6) were screened against RGS17 resulting in the identification of 131 hit compounds. Tanimoto similarity analysis was performed on all hits to allow for small scale SAR analysis of confirmed lead compounds. Follow up experiments are under way including dose response analysis and structure based analysis utilizing NMR and molecular modeling.

Support or Funding Information

Supported in part by RO1CA160470, in part by OVPRED internal funding initiative RAF‐DLR and in part by a Pre‐Doctoral Fellowship from the American Foundation of Pharmaceutical Education (AFPE)
RGS2 Modulation of Dopamine D2 Receptor Internalization and Recycling in Neuroblastoma Cells
Authors: Luessen Deborah Joyce , Hinshaw Tyler , Sun Haiguo , Marrs Glen , Chen Rong
Published: November 2023

Introduction

Altered dopamine (DA) D2 receptor (D2R) expression and function are commonly implicated in the pathophysiology of drug addiction, Parkinson's disease and schizophrenia. Internalization and recycling of D2R play a key role in the regulation of D2R activity including desensitization and resensitization. Therefore, it is imperative to improve our understanding of the molecular mechanisms underlying D2R trafficking. Regulator of G‐protein signaling (RGS) proteins are known to modulate GPCR activity by terminating G protein signaling through acceleration of GTP hydrolysis. However, recent findings highlight non‐canonical roles of RGS proteins in modulation of various aspects of GPCR including trafficking.

Methods

In the present study, we utilized a neuroblastoma (N2A) cell model to assess the role of RGS2 (regulator of G‐protein signaling 2) in the modulation of D2R trafficking and signaling by knocking down RGS2 via siRNA. Immunocytochemistry and confocal microscopy were utilized to study both constitutive and receptor agonist (quinpirole) stimulated D2R internalization and recycling. The time courses of constitutive and quinpirole (1μM) stimulated D2R internalization and recycling were evaluated at numerous time points ranging from 0 to 90 minutes.

Results

Our data indicated that RGS2 knockdown increased constitutive D2R internalization and recycling when compared to the control cells treated with scrambled siRNA. When D2R was activated by the agonist quinpirole (1 μM), there was a reduction in surface D2R level in controls cells and an increase in surface D2R level in RGS2 knockdown cells. The reduced surface D2R level in control cells induced by quinpirole treatment was due to the accelerated D2R internalization which was both clathrin and dynamin‐dependent. Both dynasore, a dynamin inhibitor, and concanavalin A, a clathrin inhibitor, blocked quinpirole‐induced D2R internalization in control cells. In contrast, RGS2 knockdown significantly attenuated quinpirole‐induced D2R internalization. Furthermore, the D2R recycling rate following quinpirole treatment in RGS2 knockdown cells was 2.5 times greater than that in the control cells. The effect of quinpirole on D2R surface trafficking was G protein dependent because pertussis toxin treatment abolished induced D2R trafficking. We also examined whether the downstream signaling molecule pERK mediates D2R internalization and recycling in the RGS2 knockdown cells. These results add a novel insight to the role of RGS2 in modulation of D2R internalization and recycling and may highlight the novel mechanistic actions of RGS2 in mediation of D2R trafficking.

Support or Funding Information

This work is supported by the National Institute On Alcohol Abuse And Alcoholism of the National Institutes of Health T32AA007565 training grant, DA006634 and a pilot grant from Center for Molecular Communication and Signaling at Wake Forest University.
Covalent Inhibition Of Acetylcholinesterase Affects Quaternary Structure As Revealed By Small Angle X‐Ray Scattering
Authors: Blumenthal Donald K , Cheng Xiaolin , Fajer Mikolai , Gerlits Oksana , Kovalevsky Andrey , Taylor Palmer , Radić Zoran
Published: November 2023

Small angle X‐ray scattering (SAXS) experiments were used to assess the influence of reversible and non‐reversible ligands on the solution structure of human acetylcholinesterase (hAChE; EC 3.1.1.7). SAXS measurements were performed using highly purified, monomeric human AChE in both non‐inhibited and organophosphate (OP) covalently inhibited states in 50 mM Tris/HCl, 100 mM NaCl, pH 7.4 at 22 °C. SAXS analysis of four OP‐hAChE conjugates (formed by VX, sarin and cyclosarin surrogates and paraoxon), as well as several reversible inhibitor‐hAChE complexes, reveals significant changes in the pair‐distance distribution function (P(r) vs r) curves in the 60 – 80 Å region for paraoxon and RP‐sarin covalent conjugates, but not for other covalent OP conjugates or reversible inhibitors of hAChE. Incubation of OP‐hAChE conjugates with the oxime reactivator antidotes 2PAM, RS‐194B, or HI‐6 resulted in reversal of the P(r) vs r profiles for paraoxon and RP‐sarin back to P(r) profiles that were similar to non‐inhibited hAChE. Analysis of the OP‐induced solution structural changes using ClusPro and FoXS indicate that “monomeric” hAChE actually consists of a reversible dimer‐monomer equilibrium ensemble that is shifted towards the monomer in paraoxon‐ and RP‐sarin‐conjugated complexes. This is the first observation of a quaternary structure modification in an AChE that results from covalent or non‐covalent binding in the AChE active center gorge. All‐atom molecular dynamics (MD) simulations at 285 K and 1 bar pressure are being used to interpret the OP‐induced conformational changes detected by SAXS. The OP‐induced changes in hAChE dimerization appear to result from distortion of the acyl pocket of the catalytic gorge that triggers changes in the dimer interface on the surface of hAChE. Analysis of OP‐induced conformational changes of hAChE by SAXS, MD simulation, and other structural methods will be used to aid the design of novel, more efficient oxime antidotes for treatment of OP intoxication.

Support or Funding Information

This work was supported by the CounterACT Program, National Institutes of Health Office of the Director, Grant 1U01NS083451 from NINDS.
Spontaneous Reactivation of Planarian Acetylcholinesterase after Inhibition by Organophosphorate Pesticides, Diazinon and Chlorpyrifos
Authors: Hirokawa Hideto , Collins Eva‐Maria , Hagstrom Danielle , Radic Zoran , Zhang Limin , Taylor Palmer
Published: November 2023

Owing to its capacity to regenerate after amputation and a dominant cholinergic influence in motor activity, we have been studying the response of the freshwater planarian, Dugesia japonica, to cholinergic inhibitors such as organophosphate pesticides. In examining cholinesterase (ChE) and other carboxylesterase activities, we find that ~85% is acetylcholinesterase (AChE) activity, with slightly different specificities to the classic reversible inhibitors, such as edrophonium and BW286c51. Of particular interest in this planarian system is the capacity of diethylphosphoryl conjugates formed from Diazinon or other pesticide exposure to reactivate spontaneously, in vitro. A similar behavior has been noted in swine butyrylcholinesterase (BChE) as noted by Brazzolotto and colleagues (Abstracts XII Int. Cholinesterase Meeting), who have suggested that an altered tertiary structure in swine BChE may predispose ChE conjugates to spontaneous reactivation. Our studies show that spontaneous reactivation in planarians can be inhibited by classical cholinesterase inhibitors and by oximes that show limited nucleophilic activity, but can be enhanced by other oximes, particularly the zwitterionic oxime, RS194B. Further, spontaneous reactivation is enhanced at low pH, in contrast to the general base catalysis by oximes on small molecule alkylphosphorate esters and methylphosphonate esters. The observed reactivation kinetics suggest unique kinetic behaviors of H2O and various nucleophiles in the active center gorge of planarian AChE, perhaps dictated by hydrogen bonding and transient electric field influences in the AChE active center gorge.

Support or Funding Information

Supported by USPHS grant GM 18360 and NS 058046.
ANTIOXIDANT AND CHEMOPREVENTIVE EFFECT OF AZADIRACHTA INDICA ON LEAD ACETATE INDUCED HEPATOTOXICITY IN RATS
Authors: Omobowale Temidayo , Oyagbemi Ademola , AdeyeOluwa Temitayo Esther , Akinboye Olajide , Adedapo Adeolu
Published: November 2023

Lead (Pb), a ubiquitous heavy metal is known to be injurious to all systems. Oxidative damage has been shown to be one of the principal mechanisms by which Pb causes toxicity. In this study, hepatic antioxidant status and markers of oxidative stress were assessed in experimental animals orally exposed to lead acetate (PbA) and was compared with groups treated with the methanolic extract of Azadirachta indica at 2 different doses.

Seventy rats were used in this study. They were divided into 7 groups each containing 10 animals. Control group (group A) received water only. Group B, C and D were dosed 0.1% lead and was withdrawn after 6 weeks. On withdrawal, Group B received water only while groups C and D were treated with 100mg/kg and 200mg/kg of the methanolic extract of Azadirachta indica respectively for 6 weeks. Similarly, groups E, F, G were given 0.2% lead orally and was withdrawn after 6 weeks. On withdrawal, group E received water only while groups F and G were treated with 100mg/kg and 200mg/kg A. indica respectively for 6 weeks.

Exposure of rats to lead acetate led to a significant (p<0.05) reduction in hepatic reduced Glutathione (GSH), Glutathione peroxidase (GPX) and Glutathione S‐ transferase. Similarly, Malondialdehyde (MDA) content increased (p<0.05) significantly in animals dosed PbA alone while there was a significant (p<0.05) reduction in rats treated with A. indica. There was significant (p<0.05) increase in nitric oxide (NO) and H2O2 concentrations in rats treated with PbA alone whereas treatment with A. indica led to significant (p<0.05) reduction in these markers of oxidative stress. Histology showed the presence of high periportal infiltration with inflammatory cells and focal necrosis in PbA treated groups but periportal infiltration was mild in Rats dosed with A. indica. In conclusion, animals exposed to PbA showed hepatotoxicity and disruption of antioxidant defence system through free radical generation and oxidative stress. Treatment of PbA exposed rats with A. indica led to significant amelioration of the hepatotoxicity induced by PbA. In conclusion, this study demonstrated the hepatoprotective effect of A. indica via antioxidant and free radical scavenging activities.

Support or Funding Information

None
THE ANTIPROLIFERATIVE AND APOPTOTIC EFFECTS OF ANNONA MURICATA EXTRACT ON PROSTATE CANCER CELLS
Authors: Bogis Ahlam , Rondon‐Ortiz Alejandro , Pino‐Figueroa Alejandro
Published: November 2023

Annona muricata (AM), a tropical evergreen tree, also known as graviola, guanabana or soursop, belongs to the custard apple trees family known as the Annonaceae. It has become known as a “cancer killer” due to its ability to reduce the proliferation of 12 different types of tumors, including breast, prostrate, lung, colon and pancreatic cancer cells. The major phytochemical compounds that have been identified in AM are cyclic hexapeptides and annonaceous acetogenins (AAGs). AAGs have been reported to have promising anticancer activities in multidrug resistant cancers (MDR). The structure of AAGs is composed of adjacent tetrahydrofuranic rings flanked by hydroxyl groups and tetrahydropyranic rings or epoxides. It has been demonstrated in previous studies that AAGs possibly inhibit the ubiquinone‐linked NADH oxidase that is constitutively over expressed in the mitochondrial membrane of cancer cells, but only transiently expressed in normal cells. The purpose of this study was to evaluate the antiproliferative and apoptotic effects of Annona muricata extract on prostate cancer cells (PC3). PC3 cells were treated with Annona muricata aqueous, methanol and ethyl acetate extracts (1–100 μg/mL). A cell viability assay utilizing 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium (MTS) was performed to evaluate the antiproliferative effects. The results demonstrated that Annona muricata ethyl acetate extract (AMEAE) produces significant reduction in cell viability (IC50 10 μg/mL). Furthermore, Western Blot analyses to test Bcl‐2 and Bax were performed onPC3 cells exposed to AMEAE. The extract showed significant reduction in the expression of the antiapoptotic protein Bcl‐2 with no effect in the expression of the proapoptotic protein Bax. Caspase 3/7 activities were also tested to verify its participation. These results suggested that the AMEAE reduces cell proliferation and/or produce cell death in PC3 cells by the induction of apoptosis. Further studies will confirm the application of this plant constituents in cancer therapy.
Novel Omega‐3 Fatty Acid Epoxygenase Metabolite Reduces Kidney Fibrosis
Authors: Sharma Amit , Khan Md. Abdul Hye , Lee Kin Sing Stephen , Hammock Bruce D. , Imig John D.
Published: November 2023

Cytochrome P450 (CYP) epoxygenase enzymes metabolize polyunsaturated fatty acid (PUFA) to epoxides. CYP epoxygenases convert omega‐3 PUFA docosahexaenoic acid (DHA) into novel epoxydocosapentaenoic (EDPs) acids that have multiple biological actions. The aim of the current study was to determine the effect of an EDP, 19,20‐EDP to reduce kidney injury in an experimental model of renal fibrosis. Mice underwent unilateral ureteral obstruction (UUO) or sham surgery and were divided into three groups; Group 1: Sham (n=6), Group 2: UUO‐Vehicle (n=6), and Group 3: UUO‐19,20‐EDP (n=6). Group 3 mice were treated with 19,20‐EDP (1mg/kg/day) administered intraperitoneally for ten days via an osmotic pump. At the end of the 10‐day experimental protocol, the kidney tissue was collected to assess effect of 19,20‐EDP on kidney fibrosis. Renal fibrosis was evaluated by Picrosirius Red stain, and α‐SMA immunostaining of kidney histological sections. Kidney biochemical analysis and mRNA expression was also conducted to evaluate hydroxyproline levels and epithelial to mesenchymal transition (EMT). Picrosirius Red collagen positive area in the UUO‐Vehicle was 5 times greater (15±1.16%, p<0.05) than the Sham group (3.3±0.5%). Mice treated with 19,20‐EDP demonstrated significantly lower collagen positive area (9.2±0.5%, p<0.05). Similarly, renal hydroxyproline content was higher in UUO‐Vehicle (0.64±0.04μg/mg tissue, p<0.05) compared to Sham (0.25±0.01 μg/mg tissue), and 19,20‐EDP treatment reduced it by 40% (0.38±0.02 μg/mg tissue). As further evidence for renal fibrosis, there was a 6‐fold increase in kidney α‐SMA in UUO‐Vehicle (2.6±0.8%, p<0.05) compared to the Sham group (0.4±0.07%). 19,20‐EDP treatment significantly decreased α‐SMA level in UUO mice (0.8±0.1%, p<0.05). Moreover, renal mRNA expression of mesenchymal markers, α‐SMA (1.3 fold), collagen IV (6.8 fold), and fibronectin (19.3 fold) were higher, and expression of the epithelial marker E‐cadherin was 1.4 fold lower in UUO‐Vehicle compared to Sham, indicating EMT in UUO. Interestingly, 19,20‐EDP reduced the renal mRNA expression of α‐SMA (52%), collagen IV (60%), and fibronectin (56%) and increased the expression of E‐cadherin (100%) in comparison to UUO‐Vehicle group. Overall, we demonstrate that a novel omega‐3 fatty acid, 19,20‐EDP, prevented UUO‐induced renal fibrosis in mice through reducing renal EMT. These results indicate an exciting opportunity for 19,20‐EDP or 19,20‐EDP mimics to effectively treat kidney fibrosis and chronic kidney disease.
Autophagy: a Novel Mechanistic Contributor to Ursodeoxycholic Acid in Alleviating Progression of Hepatic Fibrosis
Authors: Ye Hui‐Lan , Zhang Ji‐Wang , Chen Xing‐Zhou , Chen Li , Zhang Fa‐Can , Zhang Guo
Published: November 2023

Ursodeoxycholic acid (UDCA) has been found an increasingly wide utilization in the treatment of cholestatic liver fibrosis, especially for primary biliary cholangitis (PBC) and its detailed mechanisms remain unclear. Herein, this study was aimed to determine whether autophagic signaling is involved in the therapeutic effect of UDCA for liver fibrosis. Firstly, by using immunohistochemistry and fluorescent microscopic technology, autophagic molecule LC3 was found to highly express in human liver fibrotic tissues and TGFβ1 treated LX2 hepatic stellate cells(HSCs). With qRT‐PCR, autophagy promotion with rapamycin, the autophagic agonist, resulted in dramatic increment of cellular COL1A2 mRNA level, whereas blocking autophagic signaling with 3‐MA or hydroxychloroquine (HCQ) or small interference RNAs lead to decrement of COL1A2 mRNA transcription. Secondly, western blotting, qRT‐PCR and trypan blue were conducted to analyze autophagy flux, cell proliferation and collagen production in UDCA treated HSCs. UDCA treatment was found to dose‐dependently inhibit autophagy, cell proliferation as well as COL1A2 mRNA generation in TGFβ1 activated HSCs. The presence of rapamycin was shown to substantially impair the antic‐fibrotic effects of UDCA on these cells, while autophagy suppression with siRNAs targeting Atg5 or Beclin1 achieved marginally therapeutic effect. Finally, the anti‐fibrotic effect and autophagy were analyzed in CCl4‐induced liver fibrosis of rats by H&E, masson staining, qRT‐PCR and western blotting. It was found that administration of rapamycin mildly thickened fibrous septa in rats with hepatic fibrosis. However, UDCA alone or in combined with HCQ notably restored the CCl4‐induced tissue architecture disruption and collagen aggregation following inhibitory impact on autophagic signaling hallmark as LC3 IIprotein expression and P62 degradation in these rodent models. Taken together, the present study firstly revealed that UDCA displays the anti‐fibrotic role of by protecting HSC against excessive production of collagen through autophagy inhibition and provide a new insight into the pharmacological basis of UDCA treatment for hepatic fibrosis.

Support or Funding Information

National Natural Science Foundation of China, NO.30960145, NO.81360077, NO.81172260 and Guangxi Natural Science Foundation, NO.2015GXNSFDA139022, zhangguogx@hotmail.com
Pharmacologic Treatment with a Melanocortin Analogue Protects Against Pressure‐Overload Induced Cardiac Hypertrophy
Authors: Ruohonen Saku , Rinne Petteri , Ruohonen Suvi , Röyttä Matias , Savontaus Eriika
Published: November 2023

Purpose

Hypertrophic growth of the heart is a consequence of heart ability to adapt to an increased afterload and wall stress. Although left ventricular hypertrophy (LVH) may resolute with adequate therapy, this process increases the amount of irreversible cardiac fibrosis.

These morphological and functional changes in the myocardium may manifests as arrhythmias and congestive heart failure (CHF).

Melanocortins regulate important physiological functions such as energy balance and blood pressure, but their long‐term effects on cardiac remodeling remain unexplored.

In the present study, we investigated whether melanocortins affect physiological or pathologic cardiac growth and function.

Methods

First, we studied the effects of transgenic melanocortin overexpression (MSH‐OE) on physiological age‐related cardiac growth in mice. Second, we examined the effects of a stable melanocortin analogue (MT‐II) on pressure overload‐ induced cardiac hypertrophy. We subjected 8‐week‐old C57Bl/6N mice to pressure‐overload by transverse aortic constriction (TAC) and randomly assigned the mice to receive daily i.p. injections of either vehicle (physiological saline) or MT‐II (0.3 mg/kg) for 6 weeks starting 2 weeks after surgery.

Results

At the age of 6 months, MSH‐OE mice (n=11) had lower heart weight and smaller myocytes with maintained cardiac function compared to their age‐matched wild‐type littermates (n=15) while no differences in heart weight were noted at the age of 3‐months, indicating that melanocortin overactivity restrains age‐related cardiac growth. Eight weeks of pressure‐overload induced significant left ventricular (LV) hypertrophy with decreased cardiac function in saline‐treated TAC mice (n=15) compared to sham‐operated control mice (n=9).

This pathologic cardiac remodeling was attenuated in MT‐II‐treated TAC mice (n=9) (Figure 1. and 2.). Importantly, MT‐II‐treatment did not compromise cardiac function, but tended to maintain LV contractility of TAC mice. Using quantitative NMR scanning to analyze body composition, we demonstrate that MT‐II treatment also prevented the deterioration of body weight development associated with the hypertrophic disease state.

MT‐II treatment attenuated also the expression of genes regulating cardiac remodeling related mRNAs such as NppA, NppB, MMP‐2 and Col1a2.

Conclusions

Although the underlying mechanisms remain largely unclear, these results, for the first time, establish melanocortin signaling as an antihypertrophic regulator and suggest that melanocortin analogues may be potential therapeutic tools for the treatment of cardiac hypertrophy and heart failure.

Support or Funding Information

Academy of Finland, Finnish Cultural Foundation
Importance of Primates for Pharmacology Research
Authors: Shen You‐Tang , Zhang Jie , Vatner Dorothy E. , Vatner Stephen F.
Published: November 2023

The overwhelming majority of experiments in aging pathophysiology are conducted in rodent models. However, there are major species differences with humans, which can cause a major problem for drug development. One of the best examples from our laboratory was the development of beta 3 adrenergic receptors for cardiovascular function, obesity and diabetes. These drugs were effective in rodents and even some large mammalian models, but not in non‐human primates. The drugs failed in the clinics, with experience from humans, similar to the ineffectiveness in non‐human primates. Another major species difference is that rodents do not go through menopause and live for only 2–4 years. Accordingly, there are difficulties in extrapolating results in old females vs. old males to human pathophysiology. In contrast, aging female non‐human primates undergo menopause over several years, as do humans. We studied differences in left ventricular (LV) function in older primates, using the M.fascicularis model, which lives to an age in the late 20's to early 30's. There were 4 groups: young males (6.6±0.4 yrs); young females (5.5±0.5 yrs) and old males (25 ±0.3 yrs) and old females (24±0.5 yrs). As expected LV ejection fraction began to fall with aging in the males, i.e., it was significantly less, p<0.05, in old males (67±4.5%), than in young males (82±2.1%). In contrast, there were no significant differences in LV ejection fraction in old females, which were just entering menopause (73±8.3%), than in young females (79±4.6%). Thus, primates are potentially the best experimental animal model to evaluate new pharmacological therapies, particularly when related to aging and gender difference.
Pharmaceutical formulation Flavonoids and saponins extracted from black bean (Phaseolus vulgaris L.) seed coats and their hypocholesterolemic properties for the prevention of cardiovascular diseases
Authors: Jimenez‐Ramos Angelica , Chavez‐Santoscoy Rocio Alejandra
Published: November 2023

Beans exert hypolipidemic activity, and it has been attributed to flavonoids and saponins from seed coats, by attenuating lipogenesis and stimulating fatty acid oxidation and cholesterol biliary excretion through the differentially regulation of LXR probably mediated by AMPK phosphorylation. The aim of this study was to extract and quantify the major saponins and flavonoids contained in black bean seed coats and to formulate an appropriated pharmaceutical form for the delivery of previously mentioned bioactive compounds. Phytochemical extraction was performed with 90 % ethanol in acidified water with citric acid (1%). The resulting extract was concentrated and lyophilized. Phytochemicals were identified and quantified by HPLC‐UV‐ELSD. Design experiment was conducted to obtain a formulation with the best quality characteristics. Formulations were evaluated according to Mexican Official Standard NOM‐073‐SSA1‐2005 for Stability of drugs and medicines. Briefly, friability, disintegration, weight variation and hardness were measured in 5 replicates of each formulation. In vitro and in vitro assays will be performed to analyses the effects of formulation on key proteins involved in lipid metabolism and biliary cholesterol secretion.

Support or Funding Information

This research was supported by the Research Chair Funds for Full Time Professors (2014) of Autonomous University of Baja California (UABC), program Number 3888. The authors would like to acknowledge Consejo Nacional de Ciencia y Tecnología (CONACYT) in Mexico.
Protein S‐glutathionylation: the Redox Mechanism of Metabolic Stress‐induced Endothelial Barrier Dysfunction and Atherosclerosis
Authors: Han Jingyan , Shao Di , Watanabe Yosuke , Bachschmid Markus , Matsui Reiko
Published: November 2023

Background

Protein S‐glutathionylation (Pr‐SSG), the reversible formation of mixed disulfides between protein cysteinyl thiol groups and glutathione, is the most stable and abundant oxidative modification of proteins, and emerging as a critical redox signaling mechanism in cardiovascular diseases (CVD). However, its role in atherosclerosis (AS), the major cause of CVD, is not known. This study thus aimed to explore the role of Pr‐SSG in pathogenesis of AS with specific focus of vascular endothelial barrier dysfunction, an important initiating factor in the development of AS.

Methods and Results

Employing the biotin switch assay, a well‐developed approach to assess the level of reversible oxidation of proteins in cells and tissues, we found a marked increase in cysteine oxidation of aortic proteins in whole‐tissue lysates of aortas from ApoE−/− mice on Western Diet for 8 weeks, concomitant with significant decrease in reduced form of glutaredoxin‐1(Glrx‐1), a small cytosolic thiol‐transferase that can efficiently and specifically remove the GSH abducts and appears as a pivotal regulator of redox signaling cascades governing a plethora of cellular processes. Pr‐SSG in aortic tissue of ApoE−/− mice visualized by immuno‐fluorescence staining with specific antibody against protein‐GSH complex, was markedly increased in atherosclerotic lesions. Furthermore, an increased Pr‐SSG level was mainly observed in metabolic stressed aortic ECs in vivo and in vitro, which was diminished by up‐regulation of Glrx‐1 in Glrx TG/ApoE−/− mice and in Glrx adenovirus infected human aortic endothelial cells (HAECs). More importantly, the pro‐atherogenic hyper‐permeability of aortic endothelium induced by two‐week WD feeding or disturbed flow was prevented in Glrx TG/ApoE−/− mice without alternation of plasma lipid profiles and proinflammatory responses. Mechanistically, induction of Pr‐SSG in aortic endothelium disorganized cytoskeletal actin structure, while up‐regulation of Glrx‐1 strengthened aortic barrier integrity through activation of small RhoGTPase rac1 and consequent cortical actin formation. Up‐regulation of Glrx thus preserve aortic barrier function and alleviate atherosclerotic lesion development.

Conclusions

Pr‐SSG in aortic ECs is highly induced by atherogenic stimulus and plays a causative role in aortic barrier dysfunction and development of AS. S‐glutathionylation and Glrx‐1 are thus positioned as excellent target and bio‐therapeutic for cardiovascular injuries associated with oxidative stress, respectively.

Support or Funding Information

This study is supported by AHA SDG20140036 (J.H.), 1UL1TR001430 (BU CTSI), T32 HL07224, and R37 HL104017 (R.A.C.).
PARTICIPATION OF ADENYLYL CYCLASE/cAMP PATHWAY IN VASORELAXANT MECHANISM OF THE PYRANOCOUMARIN BRAYLIN IN MESENTERIC ARTERY
Authors: Albuquerque Jamile , Santos William , Jesus Rafael , Costa Rafael , Filho Otávio , Velozo Eudes , Casais e Silva Luciana , Silva Darízy
Published: November 2023

Introduction

Braylin is a pyranocoumarin isolated from the species Zanthoxylum tingoassuiba (Rutaceae). Previous studies have shown that Braylin has a possible inhibitory action in phosphodiesterase enzymes.

Aims

The aim of this study was to investigate the mechanism involved in the vasorelaxant effect of Braylin.

Methods

The experiments were performed using wistar rats (12–16 weeks/250–300g), euthanized in CO2 camera and the superior mesenteric artery were isolated and free from connective tissue and cut into rings (1–2 mm), suspended by cotton threads for isometric tension recordings in a Tyrode's solution at 37 °C, gassed with a 95% O2 and 5% CO2, under a resting tension of 0.75g. All surgical and experimental procedures were approved by the local Animal use and care ethics committee – CEUA/ICS/UFBA by the number 089/2015.

Results

The cumulative administration of Braylin (0.1–300 μM) in phenylephrine (Phe 10μM) pre‐contracted mesenteric rings, induced a concentration‐dependent vasorelaxation (Emax = 111.5 ± 7.9%, CE50 = 4.8 (3.0 – 7.7) × 10−5, n = 17), the removal of the endothelium did not change this effect (Emax = 127.6 ± 13.1%, CE50 =1.7 (4.0 – 7.3) × 10−5, n = 16). The presence of ODQ (100 μM), an inhibitor of soluble guanylyl cyclase, also did not change the effect induced by Braylin (Emax = 117.3 ± 2.6%, CE50 = 1.3 (1.9 – 8.9) × 10−5, n = 5). On the other hand, the presence of SQ 22536, an adenylyl cyclase inhibitor, reduced the relaxant effect (Emax = 115.6 ± 2.8, CE50 = 4.3 (3.0 – 6.2) × 10−5, n = 5). The incubation of the superior mesenteric rings with KT‐5720, an inhibitor of PKA, reduced the effect of Braylin (Emax = 110.4 ± 5.3%, CE50 = 2.9 (2.0 – 4.1) × 10−5, n = 5). Pre‐treatment with KCl 20 mM, TEA (1mM), BaCl2 (30μM), or 4‐aminopiridin (1mM) decreased the pharmacological potency of the Braylin significantly.

Conclusion

Those effects taken together suggest that Braylin induces vasorelaxant effects in isolated superior mesenteric artery rings from normotensive involving AC/cAMP pathway. K+ channels also seem to be important in the relaxant effect, probably BKca channels.

Support or Funding Information

Fundação de Amparo à Pesquisado Estado da Bahia (fapesb) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
The Hyaluronan Receptor RHAMM is Necessary for the Activation of Src Kinase and the Signaling Pathway for Endothelial Nitric Oxide Production
Authors: Savani Rashmin C , Liao Jie , Longoria Christopher , Wu Qian , Yuhanna Ivan S , Ullrich Victoria , Shaul Philip W , Cheong Naeun
Published: November 2023

RHAMM (Receptor for Hyaluronan‐Mediated Motility) is necessary for endothelial cell (EC) migration in vitro and angiogenesis in vivo, but the mechanisms are incompletely understood. Nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) involves a signaling cascade through Src kinase and Akt, and results in eNOS phosphorylation. A number of diverse agonists, including VEGF, HDL, estradiol, acetylcholine and insulin, activate eNOS to produce NO. We hypothesized that RHAMM is necessary for the activation of Src kinase and eNOS to produce NO. To determine the molecular mechanism by which RHAMM regulates eNOS activation, we used cultured EC and RHAMM KO mice.

RHAMM siRNA knockdown in HUVEC, BAEC or HPAEC inhibited the increased cell migration after single scratch wounding and eNOS activity observed with treatment with all EC agonists, and the exogenous NO donor NONOate rescued the migratory defect, suggesting that RHAMM is universally required for eNOS activation and NO production. RHAMM siRNA‐mediated decrease in cell migration and eNOS activity correlated with decreased Akt and eNOS phosphorylation but not Erk phosphorylation. In RHAMM KO mice, normal alveolarization and re‐endothelialization after arterial injury, processes that require NO, were delayed. RHAMM KO mice also did not respond to exogenous VEGF, but treatment with either inhaled NO (iNO) or Molsidomine normalized lung alveolarization and restored re‐endothelialization after arterial injury respectively.

Since activation of Src is a proximal step in the signaling cascade, we examined the effect of RHAMM knockdown on Src activation. RHAMM siRNA treatment of EC was associated with hyperphosphorylation of the inhibitory site of Src (Y527) and an inhibition of the phosphorylation of the activation site of Src (Y416). In this process, the phosphatase Shp2 is critical in the dephosphorylation of Y527 and Src activation, and associates with VEGFR2. Interestingly, Shp2 and Src both co‐immunoprecipitated with RHAMM in vivo as well as in vitro. Compared to EC treated with scrambled siRNA, co‐immunoprecipitation of Shp2 and VEGFR2 after VEGF stimulation was significantly reduced in RHAMM siRNA‐treated EC. These data suggest that the RHAMM‐Shp2 complex facilitates Shp2 tethering to VEGFR2, which is necessary for the dephosphorylation of Y527 in Src and the downstream activation of the signaling pathway for NO production in endothelial cells. We speculate that RHAMM is a novel target for the regulation of the wide array of physiologic and pathological functions that endothelial NO regulates.

Support or Funding Information

NIH: HL093535, HL079090, HL075930; Children's Medical Center Foundation; William Buchanan Chair in Pediatrics.
Blood‐Brain Barrier Dysfunction and Microvascular Hyperpermeability Following Mild Traumatic Brain Injury
Authors: Tharakan Binu , Alluri Himakarnika , Shaji Chinchusha Anasooya , Wiggins‐Dohlvik Katie , Beeram Madhava R , Davis Matthew L
Published: November 2023

Microvascular hyperpermeability, the excessive leakage of fluid and proteins from intravascular space to the interstitium is the leading cause of tissue vasogenic edema in a variety of pathologies. Brain edema, elevated intracranial pressure and reduced cerebral perfusion pressure occurring in traumatic brain injury (TBI) are attributed heavily to the hyperpermeability of the blood‐brain barrier (BBB). Recent studies indicate that ATP sensitive purinergic receptors (P2X7R) play a major role in promoting brain edema following traumatic brain injury. In the BBB, zonula occludens‐1 (ZO‐1) is an important TJ associated protein that binds to the transmembrane tight junction proteins (TJPs) and actin cytoskeleton intracellularly and plays a key role in maintaining its barrier functions. Our objective was to evaluate the relative difference in BBB permeability following mild and moderate TBI and to determine if P2X7R inhibition will protect the BBB against mild TBI using in vitro and in vivo approaches. For in vitro studies, rat brain microvascular endothelial cell (RBMEC) monolayers transfected with P2X7R siRNA were exposed to BzATP, a potent analogue of ATP followed by monolayer permeability assay using FITC‐dextran. The potential change in ZO‐1 protein and gene expression were studied using western blotting and RT‐PCR analysis respectively. For in vivo studies, mild and moderate TBI was induced in mice using a controlled cortical impactor and BBB integrity/permeability was studied utilizing Evans blue extravasation studied fluorometrically. To test if pharmacological inhibition of P2X7R will protect the BBB, mice pretreated with KN62 (P2X7R inhibitor) were subjected to mild TBI followed by Evans Blue assay. RBMEC monolayers exposed to BzATP (10μM; 4 hours) following P2X7R knockdown showed a decrease in permeability compared BzATP group or BzATP + control siRNA group significantly (p<0.05). The cells treated with BzATP (10μM; 4 hours) showed a decrease in ZO‐1 protein expression but the gene expression was unaltered. Mild and moderate TBI induced a significant increase in Evans blue extravasation indicating BBB hyperpermeability compared to sham control group (p<0.05). TBI group pretreated with KN62 (10 μg/gram body weight) showed a decrease in Evans blue extravasation vs. TBI group (p<0.05) suggesting BBB protection following traumatic brain injury. These results suggest that ATP‐sensitive P2X7R plays a significant role in promoting BBB hyperpermeability and pharmacological inhibition of this pathway is protective against TBI‐induced BBB dysfunction.
Endogenous tissue transglutaminase critically regulates cardiac remodeling through distinct enzymatic and non‐enzymatic actions
Authors: Shinde Arti Vijay , Dobaczewski Marcin , de Haan Judith J , Saxena Amit , Lee Kang‐Kon , Xia Ying , Chen Wei , Su Ya , Palanski Brad A , Fujikura Kana , Garcia Mario J , Melino Gerry , Frangogiannis Nikolaos G
Published: November 2023

The multifunctional protein Tissue transglutaminase (tTG) is induced in remodeling tissues and regulates cellular phenotype and matrix metabolism through enzymatic (matrix cross‐linking) effects and non‐enzymatic actions. We investigated the role of endogenous tTG in cardiac remodeling and dissected the role of enzymatic and non‐enzymatic actions in vivo and in vitro. tTG expression was upregulated in the pressure‐overloaded mouse heart and was localized in the cardiomyocytes, interstitial cells and in the extracellular matrix. tTG −/− mice had increased mortality and enhanced ventricular dilation following pressure overload, but were protected from diastolic dysfunction. In vivo, tTG loss was associated with a hypercellular cardiac interstitium, reduced collagen cross‐linking, and with accentuated matrix metalloproteinase (MMP)2 activity in the pressure‐overloaded myocardium. In contrast, inhibition of tTG activity through administration of a specific small molecule inhibitor was protective, attenuating hypertrophy, fibrosis and dysfunction. In vitro, tTG did not modulate TGF‐β‐mediated responses in cardiac fibroblasts, but exerted antiproliferative actions and, when bound to the matrix, induced synthesis of tissue inhibitor of metalloproteinases (TIMP)‐1. Following pressure overload, endogenous tTG exerts enzymatic actions that increase ventricular stiffness promoting hypertrophy and fibrosis, and protective non‐enzymatic effects that prevent chamber dilation.

Support or Funding Information

Supported by NIH grants R01 HL76246, R01 HL85440 and R01DK63158. Dr. Shinde is supported by an American Heart Association Founders’ affiliate postdoctoral grant.