bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacological Modulation of B16‐F10 Murine Melanoma‐Induced TAMs Functional Phenotype via Lipoprotein‐Based Nanoparticles
Authors: Dossou Akpedje S. , Sabnis Nirupama , Lacko Andras , Fudala Rafal
Published: May 2022

Tumor‐associated macrophages (TAMs) are one of the most abundant immune cells in the tumor microenvironment (TME). Their immunosuppressive phenotype (M2) promotes tumor progression. The re‐education of TAMs in the TME to a pro‐immune phenotype (M1) is considered to enhance the effectiveness of the anti‐tumor immune response and leads to tumor regression. An effective therapeutic approach utilizing macrophage re‐polarization will likely require the selective delivery of agents to the TME to execute the M2‐to‐M1 phenotype transformation. This strategy is known to avoid adverse immune‐related events through the utilization of nanocarriers that can effectively penetrate the TME and interact with TAMs. As a drug delivery platform, reconstituted high‐density lipoprotein nanoparticles (rHDL NPs) have attracted increased interest in cancer therapeutics, owing to their biocompatibility and their capacity to transport lipophilic drugs toward their accumulation in the tumor. We and others have shown that the scavenger receptor class B type 1 (SR‐B1), at the surface of target cells, mediates the intracellular (non‐endocytic) delivery of rHDL NPs’ payload. The novel lipophilic agonist of the STimulator of INterferon Genes (STING), MSA‐2, has been used successfully to mediate the M2‐to‐M1 reversal in murine models of cancer. In this study, we aim to deliver MSA‐2 to TAMs using mannose‐decorated rHDL NPs to induce a conversion to an M1 phenotype. Because TAMs highly express the mannose receptor CD206 that facilitates the interaction of mannose with TAMs, inducing an M1 phenotype, we anticipate a successful outcome based on this strategy. We prepared and validated the retention of the PEGylated mannose moiety on the rHDL NPs. Fluorescence anisotropy and absorbance studies showed that rHDL NPs successfully encapsulated MSA‐2. IL‐4‐induced M2 macrophages and B16‐F10 murine melanoma conditioned media‐induced TAMs showed a higher SR‐B1 expression compared to M1 macrophages. The lower uptake of the surrogate dye payload by M1 macrophages (compared to M2 macrophages and TAMs) from the mannose‐decorated rHDL NPs, corroborates the importance of the SR‐B1 receptor in rHDL payload delivery. The above findings have significant therapeutic implications for the strategy of utilizing mannose‐decorated rHDL‐NPs to selectively target TAMs in the tumor microenvironment. In addition, this formulation of MSA‐2 with the mannose‐decorated rHDL NPs can be applied in a synergistic combination with mainstream immunotherapies to improve treatment outcomes for cancer patients, diagnosed with solid tumors.
Mitochondrial‐derived ROS Contribute to the Development of Hypertensive‐Diabetic Kidney Injury
Authors: Wang Zhen , Fu Yiling , Sears Jaylan , da Silva Alexandre A. , do Carmo Jussara M. , Li Xuan , Mouton Alan , Omoto Ana Carolina M. , Hall John E.
Published: May 2022

Hypertension (HTN) is common in diabetes mellitus (DM) and substantially increases the risk for diabetic nephropathy. Current therapies only slow the progression to end‐stage renal disease (ESRD) in hypertensive‐diabetic patients rather than halt it. Thus, new insights into the mechanisms of this disease are needed.

To investigate the mechanisms by which HTN and DM interact to promote renal injury, we developed a model combining streptozotocin (STZ)‐induced type 1 DM and inter‐renal aorta constriction (AC)‐induced HTN in male Wistar rats. In this model (Wistar‐DM+AC), both kidneys are exposed to the same levels of hyperglycemia, circulating hormones, and neural influences, but the left kidney below the AC has normal to slightly reduced blood pressure (BP), while the right kidney above the AC is exposed to elevated BP. To assess the roles of mitochondrial‐derived reactive oxygen species (ROS) and mitochondrial dysfunction in causing kidney injury, Wistar‐DM+AC rats were also treated with saline vehicle or mito‐TEMPO, a mitochondrial‐targeted antioxidant, for 6 weeks starting 2 weeks after AC surgery (0.4mg/kg/day, osmotic mini‐pump). Eight weeks after AC or sham surgery, glomerular filtration rate (GFR) and urinary albumin excretion (UAE) in left and right kidneys were measured separately by collecting urine from each kidney to evaluate the change of renal function. Mitochondrial oxygen consumption rate and superoxide generation from the cortex of left and right kidneys were measured simultaneously by Oroboros high‐resolution respirometry.

Kidneys exposed to DM or HTN alone had only mild glomerular injury and slightly increased UAE. In contrast, right kidneys from Wistar‐DM+AC rats which had been exposed to DM plus HTN for 8 weeks had much greater increases in UAE (3.4±0.7 vs1.9±1.3 μg/min), significantly reduced GFR (0.5±0.1 vs 0.7±0.1 ml/min/g of kidney weight) as well as reduced mitochondrial oxygen consumption rate and increased mitochondrial ROS generation compared to left kidneys of Wistar‐DM+AC rats. In Wistar‐DM+AC rats that received mitoTEMPO treatment, the right kidneys exposed to the same levels of elevated BP and blood glucose had lower albumin excretion (1.7±0.3 μg/min), higher GFR (1.3±0.2 ml/min/g of kidney weight), and preserved mitochondrial oxygen consumption with reduced mitochondrial‐derived ROS compared to the right kidneys of vehicle‐treated Wistar‐DM+AC rats.

These results suggest that mitochondrial‐derived ROS may contribute to kidney injury when HTN is superimposed on DM. Pharmacological inhibition of mitochondrial ROS generation attenuates kidney injury and may be a potential therapeutic strategy for diabetic nephropathy.
Adult mouse kidney stem cell‐derived exosomes navigate nephron repair and regeneration in aged mice
Authors: Han Xiaobin , Akinseye Leah , Sun Zhongjie
Published: May 2022

The physiological function of kidney stem cell‐derived extracellular vesicles remains elusive. Most recently, we isolated kidney stem cells (KSCs) from adult mouse kidneys and demonstrated that KSCs‐derived exosomes normalized blood pressure and improved kidney functions in hypertensive aged mice. To study if KSCs‐derived exosomes play a role in nephron repair and regeneration, we treated aged mice with CD63‐GFP labelled KSC‐exosome (CD63/GFP KSC‐Exo) (~1.0×1011particles/mouse) and traced CD63/GFP KSC‐Exo in the kidney. We found that KSC‐exosomes were mainly detected in the proximal and distal tubular cells near the damaged glomerulus in cortex area of the aging kidney. BrdU tracing study showed that BrdU positive cells were mainly detected in the glomerulus and co‐located with Ki67 cell proliferation marker in the kidney of KSC‐exosome treated mice. KSC‐exosome treatment also increased numbers of Sca1+cells surrounding the glomerulus area of aging kidney. Interestingly, the glomerular filtration rate (GFR) was significantly increased in KSC‐exosome‐treated aged mice, suggesting improved glomerular function. To explore the mechanisms how KSC‐exosome improves kidney functions, we performed proteome analysis of KSC‐exosomes by mass spectrometry. We found several pathways that involve in the kidney development were upregulated in the KSC‐exosome from adult mice compared to that of aged mice. Notably, we further identified that FBLN2, an extracellular matrix protein, plays an important role in aging‐associated hypertension. We showed that expression of FBLN2 was downregulated in the kidney of aged mice and recombinant FBLN2 (rFBLN2) treatment normalized blood pressure and improved GFR of hypertensive aged mice. Furthermore, we found that FBLN2 is mainly located in the basement membrane of glomerulus (GBM). The thickness of GBM was increased in aged mice determined by TEM. KSC‐exosome or rFBLN2 treatment reduced the thickness of GBM, suggesting that FBLN2 may play an important role in maintaining the integrity of GBM and kidney functions. In conclusion, our finding provided the first evidence that the adult mouse kidney stem cell‐derived exosomes play an important role in nephron repair and regeneration partly via FBLN2. KSC‐exosome treatment may be a novel therapeutic strategy for hypertension‐associated kidney injuries in aging.
New insights into regulation of P‐Rex1 autoinhibition
Authors: Ravala Sandeep Kaur , Li Sheng , Chen Chun‐Liang , Cianfrocco Michael A. , Cash Jennifer N. , Tesmer John J. G.
Published: May 2022

Phosphatidylinositol 3,4,5‐trisphosphate (PIP3)–dependent Rac exchanger 1 (P‐Rex1) is a guanine nucleotide exchange factor for Rho GTPases downstream of G protein coupled receptors (GPCRs) and tyrosine kinases. It is primarily expressed in hematopoietic cells and brain. Its ectopic expression is reported in different cancers including breast, prostrate, skin, and ovarian cancers, making it a potential therapeutic target. P‐Rex1 is a multi‐domain protein consisting of a catalytic Dbl‐homology (DH) domain, followed by a PH, two DEP, two PDZ, and an inositolpolyphosphate‐4 phosphatase‐like (IP4P) domain. P‐Rex1 is regulated by PIP3, Gbg binding and phosphorylation via PKC and PKA. In unstimulated cells, P‐Rex1 is autoinhibited by intradomain interactions with its catalytic DH domain. Previously, our lab had shown that first DEP (DEP1) domain plays a key role in inhibition of DH/PH‐DEP1 exchange activity. However, there is no information available as how these domains are engaged in regulation of full length P‐Rex1. In our current study, we addressed this gap by employing single particle cryo‐electron microscopy (cryo‐EM) complemented by SAXS, HDX‐MS, and functional enzymatic assays to understand P‐Rex1 regulation. A structure with the head group of PIP3 (IP4) bound to the PH domain revealed interactions between DH and DEP1 domain that would block GTPase access to DH domain by coercing a novel configuration of the DH‐PH module. In addition, we observed that the IP4P domain was anchored to PH domain via a previously unrecognized FAT domain. Mutagenesis of residues involved in the DH–DEP1 interface led to activation, suggestive of inhibitory role of this interface. IP4 is a soluble second messenger signaling molecule which is reported to compete with PIP3 binding site in other proteins. Therefore, we tested whether it could regulate P‐Rex1 in a similar manner and found that IP4 competes with PIP3 and negatively regulates P‐Rex1 activity. Based on these observations, we propose that in unstimulated cell, P‐Rex1 is autoinhibited by intradomain interactions between the DEP1 and DH and between the FAT and PH domains, and that inositol phosphate levels may add an additional layer of desensitization by opposing P‐Rex1 membrane translocation and favoring a closed autoinhibited state.
MAL‐pharmacodynamics theory based small‐size experimental‐design and analysis, in vitro, in animals and in clinical trial, with bioinformatics algorithm, for automated computer simulation to achieve quantitative/digital/indexed conclusions
Authors: Chou Ting‐Chao
Published: May 2022

The conventional bottom‐to‐up approach of R&D is usually from aims to design, to experimental findings, to fitting empirical curves, formula, or model, to statistical analysis, to drawing conclusion/hypothesis. The alternative nonconventional, top‐to‐down approach is the mathematical system analysis to derive the unified general pharmacodynamics theory/equation/algorithm (PD) based on mass‐action law (MAL) for automated computer simulation. Thus, the approach allows using small number of dose‐data–points to fit the unified MAL‐PD theory/algorithm, for the efficient, cost‐effective automated computer simulation to achieve quantitative/digital/indexed conclusions. The MAL‐PD indicates: (I) The median‐effect eq. (MEE)“dose‐effect dynamics” defines PD‐parameters: Dm (median‐effect‐dose) for potency, and mvalue for the dynamic‐order signifying the shape of dose‐effect curve (DEC), where m=1, >1 and <1 indicate hyperbolic, sigmoidal and flat sigmoidal, respectively, and (II)Computer simulation of combination index eq. (CIE) with Fa‐CI plot, allow the quantification of Synergism (CI<1), Additive effect (CI=1) and Antagonism (CI>1) at different effect levels. The advantages of MAL‐PD approach are: (A)In animal studies or clinical trials rarely tolerated over 3 (or more) “dose‐data points” with acceptable dose‐rage and dose density; (B)MAL theory/algorithm allows the samegeneral MAL‐principle to bridge between the basic in vitro/animal studies and the clinical trials; (C)The MEE with its plot (MEP), linearizes DECs into straight lines, and thereby MEP leads to the “Two Dose‐Data‐Points Minimum Theory”, where the 3rd point is dose zero, and the 4th dose‐point is Dm, which is the universal reference‐point and dynamic‐order common‐link; (D)Since all terms of the MEE and CIE of the MAL‐PD equations are “dimensionless ratio”, therefore, MAL‐PD/CI is generally valid/applicable in R&D, regardless of nature of the effectors (e.g., drugs, biologicals, radiation, UV, pH) or units (e.g., ug, nM, IU, Rad, Gy, ug/ml, mg/kg. mg/M2), or mechanisms of action (competitive, noncompetitive, uncompetitive, sequential, ordered, ping‐pong, or random); and regardless of in vitro, in animals, or in clinical trials. In conclusion, the MAL‐PD/CI theory serves as the general largest‐common‐denominator forsimplifying the complexity and diversity of biological systems, forbiomedical R&D, and for drug evaluations, with automated simulations. Specific examples for real data analysis/simulation in vitro, and in animals will be illustrated for single drugs, and their combinations, for anticancer and antiviral studies.
Respiratory Functional and Neuroanatomical Changes in 6‐OHDA Model of Parkinson’s Disease Require Tumor Necrosis Factor Alpha Receptor 1
Authors: Cabral Laís Maria C. , Oliveira Luíz M. , dos Santos Moreira Thiago , Takakura Ana C.
Published: May 2022

Most deaths of Parkinson’s Disease (PD) patients are due to respiratory complications which appears in the late stages of this disease. Previous studies have shown brainstem degeneration which is probably linked to respiratory function impairment in a rat model of PD induced by 6‐hydroxydopamine (6‐OHDA) injection in caudate‐putamen (CPu). One of the hypotheses that explains why neurons die in this PD model is neuroinflammation mediated by a cytokine called tumor necrosis factor alpha (TNF‐α). So, this study aims to investigate the role of TNF‐α in respiratory functional and neuroanatomical deficits in the animal model of PD induced by 6‐OHDA. C57BL/6 or TNF‐α receptor 1 knockout mice (TNFR1‐/‐) were anesthetized with isoflurane 2.5% and received bilateral injection of 6‐OHDA (10 µg/µl) or vehicle in CPu through stereotaxic surgery. 20 days after surgery, mice were submitted to whole body plethysmography under normal and hypercapnic conditions (7% CO2). Animals were perfused and the brains were removed to neuroanatomical analysis. 6‐OHDA destroyed TH+ neurons in Substantia nigra (SNc) in all groups analyzed (6‐OHDA WT: 77%; 6‐OHDA TNFR1‐/‐: 79%). Degeneration was observed in preBötzinger complex (preBötC) and in retrotrapezoid nucleus (RTN) in 6‐OHDA WT group, but not in 6‐OHDA TNFR1‐/‐ group (preBötC: 6‐OHDA WT: 43%, 6‐OHDA TNFR1‐/‐: 22%; RTN: 6‐OHDA WT: 52%; 6‐OHDA TNFR1‐/‐: 15%). Astrocyte’s density in SNc is enhanced in 6‐OHDA WT group but not in 6‐OHDA TNFR1‐/‐ group (6‐OHDA WT: 122%) and is reduced in preBötC in all groups analyzed (p<0.05), while in RTN, no change was observed. Microglia analysis indicates neuroinflammation in SNc and RTN in all groups. Respiratory assessment reveals reduction in basal respiratory rate in 6‐OHDA WT group but not in 6‐OHDA TNFR1‐/‐ group (6‐OHDA WT: 25%; 6‐OHDA TNFR1‐/‐: 17%). No change was observed between groups in respiratory parameters under hypercapnia. Considering all results presented here we can state that in this model of PD, respiratory deficits are associated with neurodegeneration in respiratory brainstem areas. Abolishing TNF‐α inflammation pathway, reduction in respiratory rate and degeneration of brainstem areas involved with neural control of breathing are prevented indicating the involvement of this pathway in the respiratory changes found in the 6‐OHDA model of PD.
Effects of Dietary Nitrate Supplements on Morning Blood Pressure Surge and Sympathetic Neural Control during Orthostasis in Post‐menopausal Women
Authors: Takeda Ryosuke , Yoo Jeung‐Ki , Curtis Bryon M. , Roberts‐Reeves Monique A. , Hissen Sarah L. , Fu Qi
Published: May 2022

Post‐menopausal women are at greater risk for cardiovascular disease compared with age‐matched men. An exaggerated morning blood pressure surge (MBPS) in post‐menopausal women is well established as an independent risk factor for cardiovascular events. Dietary nitrate supplements such as nitrate‐rich beetroot juice (BRJ) may reduce blood pressure (BP), but no study has investigated the chronic effects of BRJ on MBPS and sympathetic neural control during orthostasis in post‐menopausal women. We hypothesized that dietary nitrate supplements would decrease MBPS and muscle sympathetic nerve activity (MSNA, microneurography) in post‐menopausal women.

We performed a double‐blind, placebo‐controlled, cross‐over trial and randomly assigned healthy women [n=14, 64±3 (SD) yr] to either dietary nitrate supplements (140 ml of BRJ; 800 mg nitrate/day) or placebo (140 ml of nitrate‐depleted BRJ daily) for 2 weeks each with ≥2 weeks of washout. Twenty‐four‐hour ambulatory BP monitoring was performed and MBPS was calculated as the morning systolic BP (SBP; averaged for 2 h after wake‐up) minus the lowest nocturnal SBP before and after 2‐week BRJ or placebo intervention. BP, heart rate, and MSNA were measured during 3‐min supine rest and 5‐min 60° head‐up tilt (HUT). Cardiac output (Qc; C2H2 rebreathing) and total peripheral resistance (TPR=mean BP/Qc × 80) were also measured.

MBPS decreased after BRJ intervention (Figure A; p=0.043). BRJ did not change hemodynamics or MSNA during supine rest (Figure B‐E; all, p>0.05). However, upright TPR was reduced (Figure C; p=0.007), while Qc trended greater (Figure B; p=0.072) after BRJ intervention. During HUT, MSNA burst frequency (53±15 pre vs. 48±10 bursts/min post, p=0.508 for posture × intervention interaction) and burst incidence (70±17 vs. 63±15 bursts/100 heart beats, p=0.199 for interaction) were similar, but mean burst area (Figure D; p=0.010) and total activity (Figure E; p=0.015) were lower after BRJ intervention. Placebo had no significant impact on hemodynamics and MSNA in the supine position or during HUT in post‐menopausal women (all, p<0.05).

Collectively, dietary nitrate supplements decrease morning surge in BP and upright MSNA and vasoconstriction in post‐menopausal women. Whether reductions in sympathetic activation and peripheral vasoconstriction during orthostasis contribute to the reduced MBPS in these women remains to be determined.
Mechanistic basis for the allosteric activation of mitochondrial glutaminase C, a key driver of glutamine metabolism in cancer cells
Authors: Nguyen Thuy‐Tien T. , Ramachandran Sekar , Hill Matthew J. , Cerione Richard A.
Published: May 2022

The dependence of cancer cells on glutamine metabolism, the most abundant amino acid in plasma, has been observed in many highly aggressive and deadly cancers including pancreatic cancer, triple‐negative breast cancer, and glioblastoma. The mitochondrial enzyme glutaminase C (GAC) catalyzes the hydrolysis of glutamine to glutamate, the first step in glutamine metabolism, highlighting GAC as a potentially important therapeutic target. GAC acquires maximal catalytic activity upon binding to anionic activators like inorganic phosphate. To delineate the mechanism of GAC activation, we used the tryptophan substitution of tyrosine 466 in the catalytic site of the enzyme as a fluorescence reporter for glutamine binding in the presence and absence of phosphate. We show that in the absence of phosphate, glutamine binding to the GAC (Y466W) tetramer exhibits positive cooperativity. A high‐resolution X‐ray structure of tetrameric GAC (Y466W) bound to glutamine suggests that cooperativity in substrate binding is coupled to tyrosine 249, located at the edge of the catalytic site (i.e. designated the ‘lid’), adopting two distinct conformations. In one dimer within the GAC tetramer, the lids are open and glutamine binds weakly, whereas, in the adjoining dimer, the lids are closed over the substrates resulting in higher affinity interactions. When crystallized in the presence of glutamine and phosphate, all four subunits of the GAC (Y466W) tetramer have bound glutamine with closed lids. Glutamine now binds with high affinity to each subunit, which then undergo simultaneous catalysis. These findings show how the regulated transitioning of GAC between different conformational states ensures maximal catalytic activity is reached in cancer cells only when an allosteric activator is available.
Soluble Epoxide Hydrolase Inhibitor Prevents Homocysteine‐induced Cardiac Hypertrophy via Epoxyeicosatrienoic Acid–mediated Inhibition of TRPC3 Channels
Authors: Zhou Yang , Wang Xiang‐Chong , Sun Wen‐Tao , Xue Hong‐Mei , He Guo‐Wei , Yang Qin
Published: May 2022

Objectives

Activation of soluble epoxide hydrolase (sEH), a key enzyme catalyzing the degradation of epoxyeicosatrienoic acids (EETs), has been implicated in cardiac remodeling. We previously demonstrated the involvement of sEH and transient receptor potential isoform 3 (TRPC3) channels in homocysteine‐induced hypertrophy of cardiac cells. This study investigated the anti‐hypertrophic effect of sEH inhibitor with the aim to reveal the link between sEH/EETs and TRPC3 channels in cardiac hypertrophy associated with hyperhomocysteinemia.

Materials and methods

Male Wistar rats on 10‐week standard or 2% (WT/WT) high methionine diet were given drinking water supplemented with or without the specific sEH inhibitor 1‐trifluoromethoxyphenyl‐3‐(1‐propionylpiperidin‐4‐yl) urea (TPPU, 5mg/L). In vitro, H9c2 cells were treated with homocysteine (100 μmol/L) in the presence or absence of TPPU (10 μmol/L) or 11,12‐EET (1 μmol/L). Cardiac remodeling was evaluated by echocardiography and histological analysis. Plasma homocysteine level and EETs content were determined by ELISA. Hypertrophic markers were detected by western blot and qPCR. TRPC3 channel current was recorded by patch clamp in whole‐cell mode.

Results

TPPU lowered the heart to body weight ratio, inhibited expressions of the hypertrophic markers atrial natriuretic factor (ANP) and β‐myosin heavy chain (β‐MHC), and reduced the size of cardiomyocytes in hyperhomocysteinemic rats. TPPU treatment ameliorated ejection fraction (85.50±0.87% vs. 74.18±2.19%, p<0.05) and fraction shortening (50.30±1.32% vs. 42.27±0.68%, p<0.01) of the hyperhomocysteinemic rats. Rats with hyperhomocysteinemia showed reduced levels of EETs and increased expression of TRPC3 channels, which were reversed by TPPU treatment. In vitro, TPPU and 11,12‐EET inhibited H9c2 cell enlargement induced by homocysteine, along with a downregulation of hypertrophic markers and TRPC3 channels. Homocysteine significantly increased TRPC3 channel current (4.74±0.49 vs. 1.76±0.21 pA/pF in control, p<0.001) and the enhancement was inhibited by TPPU (2.44±0.29 pA/pF, p<0.01).

Conclusions

Inactivation of sEH alleviates homocysteine‐induced cardiac hypertrophy and improves cardiac function. EET‐mediated inhibition of TRPC3 channels contributes to the protective effect of the sEH inhibitor TPPU.
A Newly Synthesized Specific PDE5a Inhibitor LW1646 Attenuated Lipid‐Induced Kidney Injuries through Inhibiting Endoplasmic Reticulum Stress
Authors: Zhao Xiaoduo , Kong Yonglun , Li Suchun , Liang Baien , Wu Deyan , Luo Haibin , Li Chunling , Wang Weidong
Published: May 2022

Hyperlipidemia plays an important role in the progression of kidney injury, likely due to renal lipotoxicity. The purpose of the present study was to investigate whether a phosphodiesterase 5a (PDE5a) inhibitor prevented lipid‐induced injury through inhibiting endoplasmic reticulum (ER) stress and apoptosis in the kidney. In human proximal tubule HK2 cells, saturated fatty acid palmitic acid (PA) treatment for 24h markedly increased protein abundance of ER stress makers BiP and CHOP, which was associated with upregulated cleaved‐caspase 3 expression and decreased ratio of Bcl2/Bax, two markers of apoptosis. A newly synthesized, specific PDE5 inhibitor LW1646 significantly decreased protein abundance of BiP, CHOP and cleaved‐caspase 3, and increased the ratio of Bcl2/Bax in HK2 cells treated by PA, likely through increasing intracellular cGMP levels. LW1646 or sildenafil, a classical inhibitor of PDE5, attenuated PA‐induced upregulation of inositol 1,4,5‐triphate receptor (IP3R) mediating calcium efflux from ER to cytoplasm, prevented PA‐induced mitochondrial depolarization, and promoted mitochondrial biogenesis in HK2 cells. Atrial natriuretic peptide (ANP) known to increase cGMP and to stimulate cGMP‐PKG signaling pathway was used to further investigate a role of cGMP in protection of PA‐induced cell injuries. ANP treatment suppressed PA‐induced ER stress, apoptosis and intracellular calcium oscillation in HK2 cells. Co‐treatment with ANP and LBQ657, a neprilysin inhibitor preventing hydrolysis of ANP, showed better protective effects than ANP treatment alone in HK2 cells treated with PA. Exogenous 8‐Br‐cGMP also attenuated PA‐induced ER stress and apoptosis. KT‐5823, a selective cGMP‐dependent PKG inhibitor, blocked protection of ANP or cGMP in HK2 cells treated with PA. Compared with controls, in mice fed with high‐fat diet for 8 weeks, the protein expression of BiP and CHOP in the kidney cortex showed 3‐fold and 2‐fold increases, respectively, which was prevented by LW1646 or sildenafil treatment. Interestingly, after high‐fat diet for 8 weeks, PDE5 knockout mice showed attenuated ER stress and apoptosis in the kidney cortex compared with wildtype mice. In conclusion, PDE5a inhibitor LW1646 inhibited lipid‐induced ER stress and apoptosis in renal tubular cells, likely via activating the cGMP‐PKG signaling pathway and maintaining mitochondrial function, it may have a therapeutic potential for preventing lipid‐induced kidney injury.
LPAR2 receptor stimulates progression of gastric cancer through β‐catenin signaling pathway
Authors: Ara Hosne , Bhattarai Susmita , Sharma Sudha , Subedi Utsab , Yu Xiuping , Bhuiyan Md. Shenuarin , Miriyala Sumitra , Panchatcharam Manikandan
Published: May 2022

Introduction

Lysophosphatidic acid (LPA) is a simple lipid that has been implicated in cellular homeostasis and pathology of different cancer by binding with G‐protein coupled receptors (LPAR1‐6). The aberrant Wnt signaling pathway has been reported to facilitate a crucial role in tumorigenesis and therapy responses. Although dysregulated β‐catenin signaling pathway has been established as a critical mediator of gastric cancer, whether LPA and LPAR2 play a role in the β ‐catenin signaling pathway during gastric cancer progression remains elusive.

Objective

Our present study explored an unknown role of LPA and LPAR2 in LPA‐induced activation of β‐catenin and its downstream signaling mechanism in β‐catenin mediated gastric cancer.

Method

We analyzed the mRNA levels of different LPA receptors in human gastric cancer by ULCAN database using TCGA dataset. LPA ELISA, Western blotting, and immunohistochemical analysis were done in the human normal stomach and cancerous stomach to measure the LPA level and check the expression of different LPA receptors. To investigate the functional role of LPAR2 in LPA‐mediated gastric cancer progression, we performed ECIS proliferation, migration assay, scratch assay, and transwell invasion assay in LPA or LPA with LPAR2 antagonist treated cells. To elucidate the molecular mechanism of LPA‐mediated gastric cancer progression, we performed Western blotting, Real‐time PCR, Luciferase assay, Immunocytochemistry, and nuclear fractionation in gastric cancer cells treated with LPA or LPA with LPAR2 antagonist.

Result

We confirmed a robust increase in LPA level and significantly increased expression of LPAR2 receptor in human gastric cancer tissue samples (P<0.001) by LPA ELISA, western blotting analysis, and immunohistochemistry, suggesting a possible role of LPA in gastric cancer through LPAR2 receptor. In cultured AGS and NCI‐N87 gastric cancer cell line, LPA treatment increased proliferation, migration, and invasion activity, whereas LPAR2 receptor antagonist abrogated the LPA induced effect. Further, when the cells were treated with LPA, it increased the β‐catenin activity, nuclear localization of β ‐catenin, and the expression of the downstream target genes of the β‐catenin signaling pathway. In addition, we confirmed LPA‐induced activation of the β‐catenin signaling pathway through the LPAR2 receptor by using LPAR2 antagonist and by knockdown of LPAR2 where LPAR2 antagonist treatment or LPAR2 knockdown rescued LPA induced activation of the β ‐catenin signaling pathway in both cell lines.

Conclusion

we established a novel LPA‐LPAR2‐β‐catenin signaling pathway in the progression of gastric cancer. All our results support that LPAR2 is a potential marker and target for developing novel therapeutic strategies in the treatment of gastric cancer.
P2Y14 receptor inhibition reverses mechanical sensitivity in a mouse model of chronic neuropathic pain
Authors: Mufti Fatma , Jung Young‐Hwan , Giancotti Luigino A. , Yu Jinha , Chen Zhoumou , Phung Ngan B. , Jacobson Kenneth A. , Salvemini Daniela
Published: May 2022

Chronic neuropathic pain is a major health issue and an economic burden that affects large numbers of people. Patients suffering from chronic pain have a significantly lowered quality of life, and to date there are no effective treatments for neuropathic pain. The P2Y14 receptor (P2Y14R) is a purinergic G‐protein coupled receptor that binds nucleotide‐sugars. P2RY14 is widely expressed in the body and is found in the immune system and nervous tissues. Few studies provide evidence that P2Y14R is involved in pain conditions. In rat traumatic nerve injury‐induced pain and post‐surgical pain models, P2RY14 transcript levels were found to increase on the same side of the spinal cord as the nerve injury. In addition, P2Y14R expression was elevated in an inflammatory pain model after a complete Freund’s adjuvant injection in the rat trigeminal ganglia. Taken together, we hypothesize that the P2Y14R plays a role in the development and maintenance of neuropathic pain. To test this, we used peripheral nerve injury model of neuropathic pain and tested several novel P2R14R antagonists, which had varying potencies and bioavailabilities. Adult male ICR mice went through unilateral chronic constriction of the sciatic nerve, and on day 7 post injury they received a P2Y14R antagonist intraperitoneally and the mechanical sensitivity in the hind paws was assessed. The antagonists rapidly (≤30 min) attenuated, some even reversed, mechanical sensitivity in the mice, with maximal effects observed typically within 1 h post‐injection, in a dose‐dependent manner. Overall, our findings provide evidence that the P2Y14R is a potential therapeutic target for treating chronic pain, and its antagonists can be candidate drugs for pain treatment.
Investigating the influence of the gut microbiome on diabetic renal disease
Authors: Xu Jiaojiao , Sanchez Jason , Gupta Kunal , Pluznick Jennifer L.
Published: May 2022

Background

Gut microbiota have been implicated in a large array of pathophysiological processes including obesity and diabetes. In this study, we utilized a diabetic renal disease model with or without oral antibiotic treatment to investigate how gut microbiota alter diabetic renal disease.

Methods

Gut microbiota biomass was depleted using a modified antibiotic treatment protocol with a mixture of 1g/L ampicillin, 1g/L neomycin, and 0.5 g/L vancomycin (modified from Rakoff‐Nahoum et al. 2004). C57BL/6J male and female mice (6‐week‐old) were randomly assigned to four different groups (n=8 per sex per group): control diet (CD), high‐fat diet (HFD), CD + antibiotics, HFD + antibiotics. The mice were treated with different diets and/or with antibiotics in drinking water for nine weeks. We measured body weight (BW), non‐fasting glucose, insulin, and glomerular filtration rate (GFR). In addition, we performed a glucose tolerance test (GTT), insulin tolerance test (ITT), and blood analysis. Finally, we did H&E staining of the kidney, and analyzed urine Na+.

Results

1) BW was significantly increased by HFD (vs CD) in both males and females regardless of antibiotic treatment. 2) Non‐fasting glucose showed no difference among these four groups in males and females. 3) Fasting glucose was dramatically elevated by HFD in males but not females (vs CD). For the GTT, both the peak glucose level and area under curve (AUC) were significantly increased in HFD vs CD, regardless of antibiotic treatment or sex. 4) For the ITT, HFD promoted insulin resistance as measured by changes in the AUC in both males and females. For males on a CD, antibiotics improved insulin responsiveness. 5) GFR was increased by HFD vs CD in both males and females, consistent with a HFD‐induced hyperfiltration, as we published previously with this same model (Halperin Kuhns, AJP Renal 2018). Intriguingly, GFR in females was also enhanced by antibiotic treatment on both the CD and HFD (week 9: CD, F: 182±12.7; HFD, F: 290±19.9, p<0.0001; CD + antibiotics, F: 282±12.6; HFD + antibiotics, F: 373±20.1; p=0.0005 CD vs. CD+antibiotics; p=0.01 HF vs. HF+antibiotics). Similarly, GFR in males also increased with antibiotic treatment in CD, but, not HFD (week 9: CD, M: 191±10.0; HFD, M: 385±19.3, p<0.0001; CD + antibiotics, M: 263±10.5; HFD + antibiotics, M: 390±32.7; p=0.04 CD vs. CD+antibiotics; p>0.99 HF vs. HF+antibiotics). Preliminary data using germ‐free (GF) mice, which do not have microbiota, show that GFR was also increased in GF males and females (vs. conventional mice with gut microbes).

Conclusion

In this study, depletion of the gut microbiota via antibiotics increased GFR in both males and females on CD. Interestingly, on a HFD, GFR increased with antibiotics in females but not males. These data suggest that gut microbial signals may directly influence renal function; we are now working to confirm these findings and to explore the underlying mechanisms.
Palisade Endings in Extraocular Muscle Exhibit Clear Motor Features but their Exact Function is still not known
Authors: Streicher Johannes , Carrero‐Rojas Genova , de la Cruz Rosa R. , Pastor Angel M. , Blumer Roland
Published: May 2022

Objective

Proprioception from extraocular muscles (EOMs) is important for proper visually‐guided behavior. Surprisingly, the classical proprioceptor pair (muscle spindles and Golgi tendon organs) is absent in the EOMs of most mammals and instead, palisade endings have been found in virtually all species analyzed so far. Due to their widespread distribution it is believed that palisade endings are candidates for EOM proprioception. We have analyzed the molecular profile and the central connection of palisade endings.

Methods

Whole mount of cat EOMs were triple labeled with neuronal markers, including markers for cholinergic axons, exocytosis proteins, and acetylcholine receptors. In anterograde tracing experiments, neuronal tracer was injected into the EOM motor nuclei.

Results

Palisade endings were formed by nerve fibers that, coming from the muscle, extended into the tendon where they made a u‐shaped turn to approach the muscle tendon junction. Recurrent nerves divided into axonal branches and terminal varicosities to form palisade endings at the tip of single muscle fibers. Molecular analyses showed that palisade endings had a cholinergic phenotype because they expressed choline acetyl transferase, the synthesizing enzyme of acetylcholine. Additionally, palisade ending expressed SNAP25, synaptobrevin, synaptotagmin, complexin, and syntaxin, all of which are key proteins involved in neurotransmitter release. Acetylcholine esterase, the degrading enzyme of acetylcholine was absent in palisade ending and no receptors for cholinergic transmission were associated with palisade endings as demonstrated by the absence of alpha‐bungarotoxin signals. Following neuronal tracer injection in the EOM motor nuclei, palisade endings were labelled.

Conclusion

Our findings show that palisade endings are cholinergic, have an exocytosis machinery for neurotransmitter (acetylcholine) release and originate from the EOM motor nuclei. However, no receptors for cholinergic transmission are associated with palisade endings suggesting that palisade ending belong to an effector system that is different from that in other skeletal muscles.
Melanin‐Concentrating Hormone (MCH) Decreases the Hypercapnic Ventilatory Response by Acting in the Lateral Hypothalamic Area (LHA)
Authors: Dias Mirela B. , Rodrigues Laísa T. C. , Gargaglioni Luciane H.
Published: May 2022

MCH plays an inhibitory role in the central hypercapnic chemoreflex, but the mechanisms were unclear so far. In this study, we tested the hypothesis that MCH1‐R receptors activation in LHA and/or Locus coeruleus (LC) is involved in the modulation of hypercapnic ventilatory response during the sleep/wake cycle. To do this, we measured pulmonary ventilation (VE), body temperature, EEG and EMG of unanesthetized male Wistar rats before and after microinjection of the MCH agonist (MCH, 0.4 mM) into the LHA and LC, or the MCH1‐R antagonist (SNAP‐94847; 63 mM), into the LHA, in room air and in 7% CO2 during wakefulness and sleep, in the dark and light periods. MCH intra‐LHA decreased the CO2 ventilatory response by 27% during wakefulness (VE = 1923 ± 384; n = 7 versus 2645 ± 293 mL kg‐1 min‐1; n = 6; P < 0.0001) and 16% during sleep (VE = 1914 ± 228; n = 6 versus2273 ± 243 mL kg‐1 min‐1; n = 4; P < 0.005) in the light, but not in the dark period while SNAP‐94847 intra‐LHA caused an increased CO2 ventilatory response during wakefulness in the light period, compared with the control group (VE = 2547 ± 277; n = 6 versus 2106 ± 329 mL kg‐1 min‐1; n = 6; P < 0.05). In the LC, MCH caused no change in the hypercapnic ventilatory response of rats. Our results suggest that MCH via MCH1‐R in the LHA, but not in the LC, exerts an inhibitory modulation of the hypercapnic ventilatory response during the light‐inactive period in rats.
Evaluation of the Antinociceptive Effects of Combining Serratiopeptidase and Ibuprofen in Acute and Chronic Nociceptive Models in Rats
Authors: Altarifi Ahmad , Abedalqader Hala , Al‐Mousa Arwa , Nuseir Khawla
Published: May 2022

Pain is a major symptom of many medical conditions with diverse etiologies. World Health Organization (WHO) indicates that 1 in 5 adults globally suffers from pain. Thus, there is a great demand on finding alternative interventions whether to be used alone or in combination with opioids or NSAIDs. In this preclinical study, we evaluated the antinociceptive effect of serratiopeptidase; a well‐known proteolytic enzyme with proven anti‐inflammatory effect and questionable analgesic effect; in the presence and absence of ibuprofen. Isobolographic analysis for drug combination was used to determine whether combination effects were synergistic. Intraperitoneal injection of 1.8 % of lactic acid and intraplantar injection of Complete Freund’s Adjuvant (CFA) were used to assess acute and chronic inflammatory pain in adult male Sprague‐Dawley rats. Both ibuprofen (10‐32 mg/kg) and serratiopeptidase (1‐3.2 mg/kg) showed a significant antinociceptive effect on lactic acid‐induced stretching. Also, only ibuprofen (32 mg/kg) showed significant increase in the number of reward bottle activations using Orofacial Pain Assessment Device (OPAD) for the assessment of acid‐depressed feeding behavior. Moreover, only ibuprofen (32 mg/kg) increased paw withdrawal threshold significantly in CFA‐induced mechanical allodynia using von Frey filaments. Combination treatments of ibuprofen and serratiopeptidase resulted in a subadditive (antagonistic) interaction on lactic acid‐stimulated body stretching behavior. However, synergistic interaction resulted by drug combination on lactic acid‐depressed feeding behavior and on CFA‐induced mechanical allodynia. In addition, the coadministration of serratiopeptidase with ibuprofen in showed a significant decrease in the duration of CFA‐induced mechanical allodynia. These data suggest that the administration of serratiopeptidase with ibuprofen may produce synergistic antinociceptive effect in chronic inflammatory conditions and pain‐depressed behaviors such as food consumption. Further clinical tests may be needed to assess this synergistic effect in human subjects.
Beta‐catenin inhibition as a novel therapeutic strategy for porphyria
Authors: Balogun Anu , Cornuet Pamela , Nejak‐Bowen Kari
Published: May 2022

Background

– The porphyrias are metabolic disorders caused by enzymatic defects in heme biosynthesis, leading to excessive accumulation of porphyrins and their precursors in the liver or bone marrow, the most active tissues in heme biosynthesis. The specific patterns of accumulation of the heme precursors aminolevulinic acid (ALA) and other intermediate compounds define clinical features of these diseases such as acute neurovisceral attacks, skin lesions, and mental changes. These debilitating diseases remain incurable, and there is an unmet need to develop effective therapies to treat them. Because the liver is either a source or sink for porphyrins, we investigated the role of Wnt/beta‐catenin pathway in this disease. Wnt/beta‐catenin signaling is an evolutionarily conserved pathway that plays an important role in liver health and pathophysiology by regulating genes important in metabolism and proliferation.

Methods

– 3,5‐Diethoxycarbonyl‐1,4‐dihydrocollidine (DDC) induces hepatic porphyria in mice and is utilized to identify signaling pathways involved in injury progression. The metabolism of DDC creates a potent inhibitor of ferrochelatase (FC), the terminal enzyme in the heme pathway, which causes the buildup of porphyrin precursors. These precursors can cause cellular abnormalities such as oxidative stress, mitochondrial dysfunction, protein aggregation and inhibition of crucial functions such as autophagy. We studied pharmacological inhibition of Wnt/beta‐catenin signaling by utilizing a beta‐catenin DsiRNA formulated into a lipid nanoparticle in mice fed DDC diet.

Results

– Our data demonstrates that mice with pharmacologically inhibited beta‐catenin have decreased liver injury due to significantly fewer porphyrin deposits. This is due to suppression of ALA‐S and ALA‐D enzymes which catalyze the first and second steps of heme biosynthesis respectively. Significantly, we identified a strong TCF4 binding site in the intron region of the Alad gene, implicating ALA‐D as a direct Wnt/beta‐catenin target. Our data, therefore, establishes a novel role of the Wnt/beta‐catenin pathway in regulating heme biosynthesis by inhibiting early steps in heme biosynthesis pathway and reducing toxic porphyrin accumulation. Furthermore, our data reveal that mice lacking beta‐catenin have increased induction of autophagy over baseline that contributes to the protection from injury by clearing accumulated toxic porphyrins.

Conclusions

– These observations collectively offer a novel opportunity to remedy porphyria by targeting the Wnt/beta‐catenin signaling pathway. Our next studies will further validate ALA‐D as the mechanistic target of beta‐catenin in the heme biosynthesis pathway. Because prior research has established crosstalk between autophagy and the Wnt signaling pathway, we will also investigate the role of beta‐catenin in regulating autophagy during porphyria.
Loss of Mitochondrial Dynamics Proteins Mitofusin‐2 and Drp‐1 in Myocardial Ischemia‐Reperfusion Injury Is Prevented by Matrix Metalloproteinase‐2 Preferring Inhibitors
Authors: Bassiouni Wesam , Seubert John M. , Schulz Richard
Published: May 2022

Objective

Matrix metalloproteinase‐2 (MMP‐2) is a ubiquitous protease that cleaves several extracellular and intracellular proteins. MMP‐2 is activated intracellularly in response to enhanced oxidative stress resulting from myocardial ischemia/reperfusion (I/R) injury. Oxidative stress impairs mitochondrial function which is regulated by different proteins including mitofusin‐2 (Mfn‐2) and dynamin‐related protein‐1 (Drp‐1) which control mitochondrial dynamics involving fusion and fission, respectively. As both proteins are localized at the mitochondrial outer membrane and MMP‐2 is localized at the mitochondrial‐endoplasmic reticulum associated membrane we hypothesized that MMP‐2 may proteolyze Mfn‐2 and Drp‐1. We therefore investigated whether inhibition of MMP‐2 could protect against the loss of mitochondrial dynamics proteins during I/R injury.

Methods

Hearts isolated from 3 month old C57BL/6J mice were perfused according to Langendorff at constant pressure and subjected to I/R injury (30 min ischemia and 40 min reperfusion) in absence or presence of MMP‐2 preferring inhibitors ARP‐100 (10 µM) or ONO‐4817 (50 µM) or their vehicle (n=5 hearts per group). Effects on mechanical function were recorded. At the end of reperfusion, the hearts were homogenized and subcellular fractions were prepared. The degradation of troponin I (TnI), an intracellular MMP‐2 target, was measured in the cytosolic fraction as a marker of MMP‐2 activity. Levels of Mfn‐2 and Drp‐1 in the mitochondrial fraction were also measured using western blot. In silico analysis of potential MMP‐2 cleavage sites was performed using Procleave.

Results

ARP‐100 or ONO‐4817 significantly increased left ventricular developed pressure compared to vehicle‐treated I/R hearts (% of pre‐ischemic baseline value at R40: IR+vehicle 25.5±3.2, IR+ARP 50.1±3.2, IR+ONO 57.3±1.6%, p<0.05). Similarly, both inhibitors significantly improved the rates of contraction and relaxation (+/‐dP/dt). TnI loss was increased in I/R hearts as shown by a significant reduction in TnI (~30 kDa) and the appearance of an ~22 kDa band. ARP‐100 or ONO‐4817 attenuated TnI loss, indicating MMP‐2 activation. Levels of Mfn‐2 and Drp‐1 in the mitochondrial fraction were significantly reduced in the I/R group and ARP‐100 or ONO‐4817 attenuated this reduction (p<0.05). Similar results were obtained using a different Mfn‐2 antibody that binds to its C‐terminus. In silico analysis of both mouse Mfn‐2 and Drp‐1 sequences showed several potential sites that could be targeted by MMP‐2.

Conclusions

During myocardial I/R injury, MMP‐2 may affect mitochondrial dynamics by proteolysis of Mfn‐2 and Drp‐1. Inhibition of MMP‐2 activity could protect against cardiac contractile dysfunction in part by preserving these proteins affecting mitochondrial fusion and fission.