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Cardiovascular Drugs and Therapy Jun 2023Diastolic dysfunction is an increasingly common cardiac pathology linked to heart failure with preserved ejection fraction. Previous studies have implicated...
PURPOSE
Diastolic dysfunction is an increasingly common cardiac pathology linked to heart failure with preserved ejection fraction. Previous studies have implicated glucagon-like peptide 1 (GLP-1) receptor agonists as potential therapies for improving diastolic dysfunction. In this study, we investigate the physiologic and metabolic changes in a mouse model of angiotensin II (AngII)-mediated diastolic dysfunction with and without the GLP-1 receptor agonist liraglutide (Lira).
METHODS
Mice were divided into sham, AngII, or AngII+Lira therapy for 4 weeks. Mice were monitored for cardiac function, weight change, and blood pressure at baseline and after 4 weeks of treatment. After 4 weeks of treatment, tissue was collected for histology, protein analysis, targeted metabolomics, and protein synthesis assays.
RESULTS
AngII treatment causes diastolic dysfunction when compared to sham mice. Lira partially prevents this dysfunction. The improvement in function in Lira mice is associated with dramatic changes in amino acid accumulation in the heart. Lira mice also have improved markers of protein translation by Western blot and increased protein synthesis by puromycin assay, suggesting that increased protein turnover protects against fibrotic remodeling and diastolic dysfunction seen in the AngII cohort. Lira mice also lost lean muscle mass compared to the AngII cohort, raising concerns about peripheral muscle scavenging as a source of the increased amino acids in the heart.
CONCLUSIONS
Lira therapy protects against AngII-mediated diastolic dysfunction, at least in part by promoting amino acid uptake and protein turnover in the heart. Liraglutide therapy is associated with loss of mean muscle mass, and long-term studies are warranted to investigate sarcopenia and frailty with liraglutide therapy in the setting of diastolic disease.
PubMed: 37382868
DOI: 10.1007/s10557-023-07482-9 -
EBioMedicine Oct 2021Nephrotic syndrome (NS) is associated with kidney podocyte injury and may occur as part of thyroid autoimmunity such as Graves' disease. Therefore, the present study was...
BACKGROUND
Nephrotic syndrome (NS) is associated with kidney podocyte injury and may occur as part of thyroid autoimmunity such as Graves' disease. Therefore, the present study was designed to ascertain if and how podocytes respond to and regulate the input of biologically active thyroid hormone (TH), 3,5,3'-triiodothyronine (T3); and also to decipher the pathophysiological role of type 3 deiodinase (D3), a membrane-bound selenoenzyme that inactivates TH, in kidney disease.
METHODS
To study D3 function in healthy and injured (PAN, puromycin aminonucleoside and LPS, Lipopolysaccharide-mediated) podocytes, immunofluorescence, qPCR and podocyte-specific D3 knockout mouse were used. Surface plasmon resonance (SPR), co-immunoprecipitation and Proximity Ligation Assay (PLA) were used for the interaction studies.
FINDINGS
Healthy podocytes expressed D3 as the predominant deiodinase isoform. Upon podocyte injury, levels of Dio3 transcript and D3 protein were dramatically reduced both in vitro and in the LPS mouse model of podocyte damage. D3 was no longer directed to the cell membrane, it accumulated in the Golgi and nucleus instead. Further, depleting D3 from the mouse podocytes resulted in foot process effacement and proteinuria. Treatment of mouse podocytes with T3 phenocopied the absence of D3 and elicited activation of αvβ3 integrin signaling, which led to podocyte injury. We also confirmed presence of an active thyroid stimulating hormone receptor (TSH-R) on mouse podocytes, engagement and activation of which resulted in podocyte injury.
INTERPRETATION
The study provided a mechanistic insight into how D3-αvβ3 integrin interaction can minimize T3-dependent integrin activation, illustrating how D3 could act as a renoprotective thyrostat in podocytes. Further, injury caused by binding of TSH-R with TSH-R antibody, as found in patients with Graves' disease, explained a plausible link between thyroid disorder and NS.
FUNDING
This work was supported by American Thyroid Association (ATA-2018-050.R1).
Topics: Animals; Cells, Cultured; Homeostasis; Humans; Integrin alphaVbeta3; Iodide Peroxidase; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Podocytes; Proteinuria; Puromycin Aminonucleoside; Receptors, Thyrotropin; Signal Transduction; Thyroid Hormones; Triiodothyronine
PubMed: 34649077
DOI: 10.1016/j.ebiom.2021.103617 -
Autophagy Jan 2024Adult stem cells are long-lived and quiescent with unique metabolic requirements. Macroautophagy/autophagy is a fundamental survival mechanism that allows cells to adapt...
Adult stem cells are long-lived and quiescent with unique metabolic requirements. Macroautophagy/autophagy is a fundamental survival mechanism that allows cells to adapt to metabolic changes by degrading and recycling intracellular components. Here we address why autophagy depletion leads to a drastic loss of the stem cell compartment. Using inducible deletion of autophagy specifically in adult hematopoietic stem cells (HSCs) and in mice chimeric for autophagy-deficient and normal HSCs, we demonstrate that the stem cell loss is cell-intrinsic. Mechanistically, autophagy-deficient HSCs showed higher expression of several amino acid transporters (AAT) when compared to autophagy-competent cells, resulting in increased amino acid (AA) uptake. This was followed by sustained MTOR (mechanistic target of rapamycin) activation, with enlarged cell size, glucose uptake and translation, which is detrimental to the quiescent HSCs. MTOR inhibition by rapamycin treatment was able to rescue autophagy-deficient HSC loss and bone marrow failure and resulted in better reconstitution after transplantation. Our results suggest that targeting MTOR may improve aged stem cell function, promote reprogramming and stem cell transplantation. 5FU: fluoracil; AA: amino acids; AKT/PKB: thymoma viral proto-oncogene 1; ATF4: activating transcription factor 4; BafA: bafilomycin A; BM: bone marrow; EIF2: eukaryotic initiation factor 2; EIF4EBP1/4EBP1: eukaryotic translation initiation factor 4E binding protein 1; KIT/CD117/c-Kit: KIT proto-oncogene receptor tyrosine kinase; HSCs: hematopoietic stem cells; HSPCs: hematopoietic stem and progenitor cells; Kyn: kynurenine; LSK: lineage (Lin), LY6A/Sca-1, KIT/c-Kit/CD117; LY6A/Sca-1: lymphocyte antigen 6 family member A; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; MTORC2: MTOR complex 2; OPP: O-propargyl-puromycin; PI3K: phosphoinositide 3-kinase; poly(I:C): polyinosinic:polycytidylic acid; RPS6/S6: ribosomal protein S6; tam: tamoxifen; TCA: tricarboxylic acid; TFEB: transcription factor EB; PTPRC/CD45: Protein Tyrosine Phosphatase Receptor Type C, CD45 antigen.
Topics: Mice; Animals; Mechanistic Target of Rapamycin Complex 1; Signal Transduction; Autophagy; Phosphatidylinositol 3-Kinases; Hematopoietic Stem Cells; Mechanistic Target of Rapamycin Complex 2; Sirolimus
PubMed: 37614038
DOI: 10.1080/15548627.2023.2247310 -
Fluids and Barriers of the CNS Feb 2023Brain microvascular endothelial cells (BMECs) play a major role in the blood-brain barrier (BBB), and are critical for establishing an in vitro BBB model. Currently,...
BACKGROUND
Brain microvascular endothelial cells (BMECs) play a major role in the blood-brain barrier (BBB), and are critical for establishing an in vitro BBB model. Currently, iPSC-derived BMECs (iBMECs) have been used to construct in vitro BBB models with physiological barrier functions, such as high trans-endothelial electrical resistance (TEER) and expression of transporter proteins. However, the relatively low p-glycoprotein (P-gp) level and a decrease in the efflux ratio of its substrates in iBMECs suggest their immature nature. Therefore, more mature iBMECs by optimizing the differentiation induction protocol is beneficial for establishing a more reliable in vitro BBB model for studying central nervous system (CNS) drug transport.
METHODS
To identify human brain endothelial cell fate-inducing factors, HUVEC was transfected with Zic3A-, Zic3B-, and Sox18-expressing lentivirus vector. Since SOX18 was found to induce BMEC properties, we used a Dox-inducible Tet-on system to express SOX18 during iBMEC differentiation and explored the impact of SOX18 expression on iBMEC maturation.
RESULTS
Sox18-mediated iBMECs achieved a higher TEER value than normal iBMECs (> 3000 Ω cm). From day 6 to day 10 (d6-10 group), the iBMECs with SOX18 expression expressed a series of tight junction markers and showed upregulation of Mfsd2a, a specific marker of the BBB. The d6-10 group also expressed SLC2A1/Glut1 at levels as high as normal iBMECs, and upregulated ABCB1/P-gp and ABCC1/MRP1 expression. Moreover, Sox18-mediated iBMECs showed higher viability than normal iBMECs after puromycin treatment, indicating that SOX18 expression could upregulate P-gp activity in iBMECs.
CONCLUSIONS
Inducible SOX18 expression in iBMECs gained BBB phenotypes, including high TEER values and upregulation of tight junction-related genes, endothelial cell (EC) markers, BBB transporters, and higher cell viability after treatment with puromycin. Collectively, we provide a differentiation method for the maturation of human iPS cell-derived BMECs with SOX18 expression, describing its contribution to form an in vitro BBB model for CNS drug transport studies.
Topics: Humans; Endothelial Cells; Induced Pluripotent Stem Cells; Cells, Cultured; Brain; Blood-Brain Barrier; SOXF Transcription Factors
PubMed: 36732767
DOI: 10.1186/s12987-023-00408-5 -
Cells Aug 2023Podocyte cellular injury and detachment from glomerular capillaries constitute a critical factor contributing to kidney disease. Notably, transcription factors are...
Podocyte cellular injury and detachment from glomerular capillaries constitute a critical factor contributing to kidney disease. Notably, transcription factors are instrumental in maintaining podocyte differentiation and homeostasis. This study explores the hitherto uninvestigated expression of Nuclear Factor Erythroid 2-related Factor 1 (NFE2L1) in podocytes. We evaluated the podocyte expression of NFE2L1, Nuclear Factor Erythroid 2-related Factor 2 (NFE2L2), and NAD(P)H:quinone Oxidoreductase (NQO1) in 127 human glomerular disease biopsies using multiplexed immunofluorescence and image analysis. We found that both NFE2L1 and NQO1 expressions were significantly diminished across all observed renal diseases. Furthermore, we exposed human immortalized podocytes and ex vivo kidney slices to Puromycin Aminonucleoside (PAN) and characterized the NFE2L1 protein isoform expression. PAN treatment led to a reduction in the nuclear expression of NFE2L1 in ex vivo kidney slices and podocytes.
Topics: Humans; Podocytes; Gene Expression Regulation; Kidney Diseases; Kidney; Kidney Glomerulus; NAD; NF-E2-Related Factor 1
PubMed: 37681897
DOI: 10.3390/cells12172165 -
Journal of Molecular Biology Apr 2022Biological protein synthesis is mediated by the ribosome, and employs ~20 proteinogenic amino acids as building blocks. Through the use of misacylated tRNAs, presently... (Review)
Review
Biological protein synthesis is mediated by the ribosome, and employs ~20 proteinogenic amino acids as building blocks. Through the use of misacylated tRNAs, presently accessible by any of several strategies, it is now possible to employ in vitro and in vivo protein biosynthesis to elaborate proteins containing a much larger variety of amino acid building blocks. However, the incorporation of this broader variety of amino acids is limited to those species utilized by the ribosome. As a consequence, virtually all of the substrates utilized over time have been L-α-amino acids. In recent years, a variety of structural and biochemical studies have provided important insights into those regions of the 23S ribosomal RNA that are involved in peptide bond formation. Subsequent experiments, involving the randomization of key regions of 23S rRNA required for peptide bond formation, have afforded libraries of E. coli harboring plasmids with the rrnB gene modified in the key regions. Selections based on the use of modified puromycin derivatives with altered amino acids then identified clones uniquely sensitive to individual puromycin derivatives. These clones often recognized misacylated tRNAs containing altered amino acids similar to those in the modified puromycins, and incorporated the amino acid analogues into proteins. In this fashion, it has been possible to realize the synthesis of proteins containing D-amino acids, β-amino acids, phosphorylated amino acids, as well as long chain and cyclic amino acids in which the nucleophilic amino group is not in the α-position. Of special interest have been dipeptides and dipeptidomimetics of diverse utility.
Topics: Amino Acids; Dipeptides; Escherichia coli; Genetic Code; Protein Biosynthesis; Proteins; Puromycin; RNA, Ribosomal, 23S; RNA, Transfer; Ribosomes
PubMed: 34419431
DOI: 10.1016/j.jmb.2021.167211 -
International Journal of Medical... 2022Podocytes are specialized cells of the glomerulus that play important structural and functional roles in maintaining the filtration barrier. Loss and injury of podocytes...
Podocytes are specialized cells of the glomerulus that play important structural and functional roles in maintaining the filtration barrier. Loss and injury of podocytes are leading factors of glomerular disease and kidney failure. Recent studies found that phosphatase and tensin homolog (PTEN) may play a critical role in maintaining the normal structure and function in podocytes. However, we still understand very little about how PTEN is regulated under podocyte injury conditions. In this study, We therefore investigated whether PTEN could play a role in podocyte injury induced by puromycin aminonucleoside (PAN), and whether dexamethasone (DEX) alleviates podocyte injury by PTEN/PI3K/Akt signaling. Our results showed that PI3K/Akt pathway was activated in podocytes exposed to PAN conditions, accompanied by down-regulation of the PTEN and microtubule-associated light chain 3 (LC3) expression.podocyte-specific knockout of PTEN significantly promoted podocyte injury, The potential renoprotection of overexpressed PTEN in podocytes was partly attributed with an improvement in autophagy and the inhibition of apoptosis.These novel findings also suggest that targeting PTEN might be a novel and promising therapeutic strategy against podocyte injury.
Topics: Autophagy; Phosphatidylinositol 3-Kinases; Podocytes; Proto-Oncogene Proteins c-akt; Puromycin Aminonucleoside
PubMed: 36035365
DOI: 10.7150/ijms.72988 -
American Journal of Nephrology 2021Synbindin, originally identified as a neuronal cytoplasmic molecule, was found in glomeruli. The cDNA subtractive hybridization technique showed the mRNA expression of...
INTRODUCTION
Synbindin, originally identified as a neuronal cytoplasmic molecule, was found in glomeruli. The cDNA subtractive hybridization technique showed the mRNA expression of synbindin in glomeruli was downregulated in puromycin aminonucleoside (PAN) nephropathy, a mimic of minimal-change nephrotic syndrome.
METHODS
The expression of synbindin in podocytes was analyzed in normal rats and 2 types of rat nephrotic models, anti-nephrin antibody-induced nephropathy, a pure slit diaphragm injury model, and PAN nephropathy, by immunohistochemical analysis and RT-PCR techniques. To elucidate the function of synbindin, a gene silencing study with human cultured podocytes was performed.
RESULTS
Synbindin was mainly expressed at the slit diaphragm area of glomerular epithelial cells (podocytes). In both nephrotic models, decreased mRNA expression and the altered staining of synbindin were already detected at the early phase when proteinuria and the altered staining of nephrin, a key molecule of slit diaphragm, were not detected yet. Synbindin staining was clearly reduced when severe proteinuria was observed. When the cultured podocytes were treated with siRNA for synbindin, the cell changed to a round shape, and filamentous actin structure was clearly altered. The expression of ephrin-B1, a transmembrane protein at slit diaphragm, was clearly lowered, and synaptic vesicle-associated protein 2B (SV2B) was upregulated in the synbindin knockdown cells.
CONCLUSION
Synbindin participates in maintaining foot processes and slit diaphragm as a downstream molecule of SV2B-mediated vesicle transport. Synbindin downregulation participates in slit diaphragm dysfunction. Synbindin can be an early marker to detect podocyte injury.
Topics: Animals; Female; Rats; Down-Regulation; Kidney Diseases; Kidney Glomerulus; Podocytes; Rats, Wistar
PubMed: 34515036
DOI: 10.1159/000517975 -
Methods in Molecular Biology (Clifton,... 2024Mesenchymal stem cells (MSC) are multipotent stem cells that display the capacity to generate the tissue in which they reside. MSC have been used as progenitor cells to...
Mesenchymal stem cells (MSC) are multipotent stem cells that display the capacity to generate the tissue in which they reside. MSC have been used as progenitor cells to engineer cartilage implants that can be used to repair chondral and osteochondral lesions, or as trophic producers of bioactive factors to initiate endogenous regenerative activities in the arthritic joint. Targeted gene therapy might further enhance the capacity of MSC for chondrogenesis. By using a clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins genomic manipulation technique, target gene-modified MSC would be a promising therapeutic option for regeneration of diseased joints in the treatment of RA.
Topics: Mesenchymal Stem Cells; Multipotent Stem Cells; Stem Cells; CRISPR-Associated Proteins; Chondrogenesis
PubMed: 38270877
DOI: 10.1007/978-1-0716-3682-4_18 -
Bio-protocol Aug 2023Ribosome footprint profiling has demonstrated that ribosomes can be slowed or stalled on select mRNAs, often due to the presence of rare codons, stalling motifs, or via...
Ribosome footprint profiling has demonstrated that ribosomes can be slowed or stalled on select mRNAs, often due to the presence of rare codons, stalling motifs, or via a ribosome-binding protein (e.g., FMRP). Stalled ribosomes can act as physical roadblocks for trailing ribosomes and ultimately can cause ribosome collisions that stimulate no-go mRNA decay. Detecting stalled or slowed ribosomes in cells by ribosome footprint profiling or classic polysome profiling is laborious, technically challenging, and low throughput. Here, we present a protocol to assay for stalled ribosomes on in vitro-transcribed reporter mRNAs using a robust, commercially available mammalian in vitro translation lysate and an optimized low-speed sucrose cushion. In short, we take advantage of the ability of puromycin to incorporate into the nascent polypeptide and cause the ribosome to dissociate from the mRNA during active elongation, as well as the ability to selectively pellet ribosomes through a low-speed sucrose cushion due to their large molecular weight. Stalled ribosomes are not actively elongating and do not incorporate puromycin, allowing the ribosome-bound mRNA to pellet in the low-speed sucrose cushion. RT-qPCR is used to quantify the amount of ribosome-bound reporter mRNA in the pellet. This workflow allows for direct assessment of stalled ribosomes and is fully amendable to insertion of putative stalling motifs in the target mRNA, as well as supplementation with recombinant proteins or small molecule inhibitors that target translation elongation. Key features This protocol is optimized for cap-dependent in vitro translation in the dynamic linear range. Details for generating capped reporter mRNA in one day are provided. Requires as little as one day to complete if starting with in vitro-transcribed mRNA. This protocol requires access to an ultracentrifuge and a real-time PCR system.
PubMed: 37638299
DOI: 10.21769/BioProtoc.4744