The extract of Ilex kudingcha inhibits atherosclerosis in apoE-deficient mice by suppressing cholesterol accumulation in macrophages

2021 ◽  
Author(s):  
Yukio Fujiwara ◽  
Shota Okada ◽  
Keisuke Uryu ◽  
Isafumi Maru ◽  
Yoshihiro Komohara
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Human Monocyte ◽  
Inhibitory Effects ◽  
Cholesterol Accumulation ◽  
Cholesterol Acyl Transferase ◽  
Ovary Cells ◽  
Acat Activity ◽  
Atherosclerotic Lesions ◽  
Deficient Mice

Abstract It was previously reported that oleanolic acid and ursolic acid, triterpenoid compounds occurring in Ilex kudingcha, ameliorate hyperlipidemia and atherosclerosis in apoE-deficient mice. In the present study, we investigated whether Ilex kudingcha extract exerts similar inhibitory effects on cholesterol accumulation in human monocyte-derived macrophages (HMDMs) and atherogenesis in apoE-deficient mice. Ilex kudingcha extract significantly inhibited cholesterol ester (CE) accumulation induced by acetyl-LDL (acetylated LDL) in HMDMs; however, it generated no effect on cell viability in HMDMs. Ilex kudingcha extract also suppressed CE accumulation in acyl-CoA: cholesterol acyl-transferase (ACAT)-overexpressing CHO (Chinese hamster ovary) cells, thereby indicating that it inhibits ACAT activity. Furthermore, the oral administration of Ilex kudingcha extract to apoE-deficient mice significantly decreased the levels of serum cholesterol, triglyceride, sLOX-1, as well as the regions of atherosclerotic lesions in the mice. Our study reveals crucial new-found evidence that Ilex kudingcha extract significantly inhibits ACAT activity and suppresses atherogenesis.

scholarly journals The class I scavenger receptor CD163 promotes internalization of ADAMTS13 by macrophages

2017 ◽  
Vol 1 (5) ◽  
pp. 293-305 ◽  
Author(s):  
Fabian C. Verbij ◽  
Nicoletta Sorvillo ◽  
Paul H. P. Kaijen ◽  
Johana Hrdinova ◽  
Ivan Peyron ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Scavenger Receptor ◽  
Chinese Hamster ◽  
Human Monocyte ◽  
Mannose Receptor ◽  
Class I ◽  
Spectrometric Analysis ◽  
Ovary Cells ◽  
Macrophage Mannose Receptor ◽  
Partial Blocking

Abstract Internalization of ADAMTS13 by macrophages may contribute to its clearance from the circulation. Here we investigated endocytic mechanisms that contribute to the uptake of ADAMTS13 by macrophages. Human monocyte-derived macrophages were used to monitor the uptake of fluorescently labeled recombinant ADAMTS13 by flow cytometry. Internalization of ADAMTS13 was blocked upon addition of the cell-permeable dynamin inhibitor dynasore. Partial blocking of ADAMTS13 uptake was observed by using mannan; however, uptake was not affected by an antibody that blocked binding to the macrophage mannose receptor CD206, which suggests that other endocytic receptors contribute to the internalization of ADAMTS13 by macrophages. A pull-down with ADAMTS13 and subsequent mass spectrometric analysis identified the class I scavenger receptor CD163 as a candidate receptor for ADAMTS13. Blocking experiments with monoclonal anti-CD163 antibody EDHu-1 resulted in decreased ADAMTS13 internalization by macrophages. Pronounced inhibition of ADAMTS13 uptake by EDHu-1 was observed in CD163 high-expressing macrophages. In agreement with these findings, CD163-expressing Chinese hamster ovary cells were capable of rapidly internalizing ADAMTS13. Surface plasmon resonance revealed binding of ADAMTS13 to scavenger receptor cysteine-rich domains 1-9 and 1-5 of CD163. Taken together, our data identify CD163 as a major endocytic receptor for ADAMTS13 on macrophages.

scholarly journals Human Acyl-Coenzyme A:Cholesterol Acyltransferase 1 (acat1) Sequences Located in Two Different Chromosomes (7 and 1) Are Required to Produce a Novel ACAT1 Isoenzyme with Additional Sequence at the N Terminus

2004 ◽  
Vol 279 (44) ◽  
pp. 46253-46262 ◽  
Author(s):  
Li Yang ◽  
Oneil Lee ◽  
Jia Chen ◽  
Jiang Chen ◽  
Catherine C. Y. Chang ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Human Monocyte ◽  
Functional Protein ◽  
Rare Form ◽  
Ovary Cells ◽  
Sds Page ◽  
Translation Initiation Codon ◽  
Additional Sequence ◽  
Additional Amino Acid

A rare form of human ACAT1 mRNA, containing the optional long 5′-untranslated region, is produced as a 4.3-kelonucleotide chimeric mRNA through a novel interchromosomaltrans-splicing of two discontinuous RNAs transcribed from chromosomes 1 and 7 (Li, B. L., Li, X. L., Duan, Z. J., Lee, O., Lin, S., Ma, Z. M., Chang, C. C., Yang, X. Y., Park, J. P., Mohandas, T. K., Noll, W., Chan, L., and Chang, T. Y. (1999)J. Biol. Chem.274, 11060–11071). To investigate its function, we express the chimeric ACAT1 mRNA in Chinese hamster ovary cells and show that it can produce a larger ACAT1 protein, with an apparent molecular mass of 56 kDa on SDS-PAGE, in addition to the normal, 50-kDa ACAT1 protein, which is produced from the ACAT1 mRNAs without the optional long 5′-untranslated repeat. To produce the 56-kDa ACAT1,acat1sequences located at both chromosomes 7 and 1 are required. The 56-kDa ACAT1 can be recognized by specific antibodies prepared against the predicted additional amino acid sequence located upstream of the N-terminal of the ACAT1ORF. The translation initiation codon for the 56-kDa protein is GGC, which encodes for glycine, as deduced by mutation analysis and mass spectrometry. Similar to the 50-kDa protein, when expressed alone, the 56-kDa ACAT1 is located in the endoplasmic reticulum and is enzymatically active. The 56-kDa ACAT1 is present in native human cells, including human monocyte-derived macrophages. Our current results show that the function of the chimeric ACAT1 mRNA is to increase the ACAT enzyme diversity by producing a novel isoenzyme. To our knowledge, our result provides the first mammalian example that a trans-spliced mRNA produces a functional protein.

scholarly journals The murine macrophage apoB-48 receptor gene (Apob-48r)

2002 ◽  
Vol 43 (8) ◽  
pp. 1181-1191 ◽  
Author(s):  
Matthew L. Brown ◽  
Katsumasa Yui ◽  
Jonathan D. Smith ◽  
Renée C. LeBoeuf ◽  
Wei Weng ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Attachment Site ◽  
Foam Cells ◽  
Human Macrophage ◽  
Murine Macrophage ◽  
Hydrophobic Domain ◽  
Atherosclerotic Lesions ◽  
Deficient Mice

Previously we cloned the human macrophage apolipoprotein B-48 receptor (ApoB-48R) and documented its expression in human atherosclerotic foam cells (1). Now we have identified and characterized the murine macrophage apob-48r cDNA gene sequence and its chromasomal location. The cDNA (3,615 bp) -deduced amino acid (aa) sequence (942 aa) is ∼45% identical to the human macrophage APOB-48R, but not to other known gene families. The murine Apob-48r gene, like the human APOB-48R gene, consists of four exons interrupted by three small introns and is syntenically located on chromosome 7. Functionally significant conserved domains include an N-terminal hydrophobic domain, a glycosaminoglycan attachment site, an N-glycosylation site, and an ExxxLL internalization motif C-terminal to the putative internal transmembrane domain. Two conserved coiled-coil domains are likely involved in the spontaneous homodimerization that generates the active dimeric ligand binding species (mouse, ∼190 kDa; human, ∼200 kDa). Transfection of the murine apoB-48R into Chinese hamster ovary cells (CHOs) confers apoB-48R function: rapid, high-affinity, specific uptake of known triglyceride-rich lipoprotein ligands of the apoB-48R and, of note, uptake of the cholesteryl ester-rich apoB-48-containing very low density lipoproteins that accumulate in atherosclerosis-prone apoE-deficient mice. Uptake of these ligands by murine apoB-48R-transfected CHOs causes saturable, visible cellular triglyceride and cholesterol accumulation in vitro that resemble foam cells of atherosclerotic lesions.In aggregate, the data presented here and that previously published suggest that the apoE-independent murine apoB-48R pathway may contribute to the spontaneous development of atherosclerotic lesions rich in macrophage-derived foam cells observed in apoE-deficient mice, a murine model of human atherosclerosis.

scholarly journals Phosphorylation of IGFBP-1 at Discrete Sites Elicits Variable Effects on IGF-I Receptor Autophosphorylation

Endocrinology ◽  
2013 ◽  
Vol 154 (3) ◽  
pp. 1130-1143 ◽  
Author(s):  
Majida Abu Shehab ◽  
Cristiana Iosef ◽  
Robert Wildgruber ◽  
Girish Sardana ◽  
Madhulika B. Gupta
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Phosphorylation Sites ◽  
Wild Type ◽  
Inhibitory Effects ◽  
Ovary Cells ◽  
Igf I ◽  
Igf Binding ◽  
Igf Binding Protein

Abstract We previously demonstrated that hypoxia and leucine deprivation cause hyperphosphorylation of IGF-binding protein-1 (IGFBP-1) at discrete sites that markedly enhanced IGF-I affinity and inhibited IGF-I-stimulated cell growth. In this study we investigated the functional role of these phosphorylation sites using mutagenesis. We created three IGFBP-1 mutants in which individual serine (S119/S169/S98) residues were substituted with alanine and S101A was recreated for comparison. The wild-type (WT) and mutant IGFBP-1 were expressed in Chinese hamster ovary cells and IGFBP-1 in cell media was isolated using isoelectric-focusing-free-flow electrophoresis. BIACore analysis indicated that the changes in IGF-I affinity for S98A and S169A were moderate, whereas S119A greatly reduced the affinity of IGFBP-1 for IGF-I (100-fold, P < .0001). Similar results were obtained with S101A. The IGF-I affinity changes of the mutants were reflected in their ability to inhibit IGF-I-induced receptor autophosphorylation. Employing receptor-stimulation assay using IGF-IR-overexpressing P6 cells, we found that WT-IGFBP-1 inhibited IGF-IRβ autophosphorylation (∼2-fold, P < .001), possibly attributable to sequestration of IGF-I. Relative to WT, S98A and S169A mutants did not inhibit receptor autophosphorylation. S119A, on the other hand, greatly stimulated the receptor (2.3-fold, P < .05). The data with S101A matched S119A. In summary, we show that phosphorylation at S98 and S169 resulted in milder changes in IGF-I action; nonetheless most dramatic inhibitory effects on the biological activity of IGF-I were due to IGFBP-1 phosphorylation at S119. Our results provide novel demonstration that IGFBP-1 phosphorylation at S119 can enhance affinity for IGF-I possibly through stabilization of the IGF-IGFBP-1 complex. These data also propose that the synergistic interaction of distinct phosphorylation sites may be important in eliciting more pronounced effects on IGF-I affinity that needs further investigation.

Recombinant Human Soluble Thrombomodulin Delivers Bounded Thrombin to Antithrombin III: Thrombomodulin Associates with Free Thrombin and Is Recycled to Activate Protein C

1993 ◽  
Vol 70 (03) ◽  
pp. 418-422 ◽  
Author(s):  
Masaharu Aritomi ◽  
Naoko Watanabe ◽  
Rika Ohishi ◽  
Komakazu Gomi ◽  
Takao Kiyota ◽  
...  
Keyword(s):  
Protein C ◽  
Antithrombin Iii ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Time Lag ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Soluble Thrombomodulin ◽  
Simulation Based ◽  
Recombinant Human Soluble Thrombomodulin

SummaryRecombinant human soluble thrombomodulin (rhs-TM), having no transmembrane domain or chondroitin sulfate, was expressed in Chinese hamster ovary cells. Interactions between rhs-TM, thrombin (Th), protein C (PC) and antithrombin III (ATIII) were studied. Equilibrium between rhs-TM and Th had no detectable time lag in clotting inhibition (K d = 26 nM) or PC activation (K d = 22 nM), while ATIII inhibited Th at a bimolecular rate constant = 5,200 M-1s-1 (K d <0.2 nM). A mixture of ATIII, Th and rhs-TM showed that ATIII reacted with Th slower than rhs-TM, whose presence did not affect the reaction between ATIII and Th. In a mixture of rhs-TM, ATIII and PC, the repeated addition of Th caused the repeated activation of PC; which was consistent with the Simulation based on the assumption that rhs-TM is recycled as a Th cofactor. From these results, we concluded that upon inhibition of the rhs-TM-Th complex by ATIII, rhs-TM is released to recombine with free Th and begins to activate PC, while the Th-ATIII complex does not affect rhs-TM-Th equilibrium.

Malignancy-related characteristics of wild type and drug-resistant chinese hamster ovary cells

Pathology ◽  
1993 ◽  
Vol 25 (3) ◽  
pp. 268-276 ◽  
Author(s):  
Wanda B. Mackinnon ◽  
Marlen Dyne ◽  
Rebecca Hancock ◽  
Carolyn E. Mountford ◽  
Adrienne J. Grant ◽  
...  
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Drug Resistant ◽  
Wild Type ◽  
Ovary Cells

Review of the current methods of Chinese Hamster Ovary (CHO) cells cultivation for production of therapeutic protein

2020 ◽  
Vol 17 ◽  
Author(s):  
Shazid Md. Sharker ◽  
Md. Atiqur Rahman
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Mass Production ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Therapeutic Protein ◽  
Specific Productivity ◽  
Ovary Cells ◽  
Further Development ◽  
Interesting Area

Most of clinical approved protein-based drugs or under in clinical trial have a profound impact in the treatment of critical diseases. The mammalian eukaryotic cells culture approaches, particularly the CHO (Chinese Hamster Ovary) cells are mainly used in the biopharmaceutical industry for the mass-production of therapeutic protein. Recent advances in CHO cell bioprocessing to yield recombinant proteins and monoclonal antibodies have enabled the expression of quality protein. The developments of cell lines are possible to upgrade specific productivity. As a result, it holds an interesting area for academic as well as industrial researchers around the world. This review will concentrate on the recent progress of the mammalian CHO cells culture technology and the future scope of further development for the mass-production of protein therapeutics.

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