Syndecan-1 Facilitates the Human Mesenchymal Stem Cell Osteo-Adipogenic Balance

Bone marrow-derived human mesenchymal stems cells (hMSCs) are precursors to adipocyte and osteoblast lineage cells. Dysregulation of the osteo-adipogenic balance has been implicated in pathological conditions involving bone loss. Heparan sulfate proteoglycans (HSPGs) such as cell membrane-bound syndecans (SDCs) and glypicans (GPCs) mediate hMSC lineage differentiation and with syndecan-1 (SDC-1) reported in both adipogenesis and osteogenesis, these macromolecules are potential regulators of the osteo-adipogenic balance. Here, we disrupted the HSPG profile in primary hMSC cultures via temporal knockdown (KD) of SDC-1 using RNA interference (RNAi) in undifferentiated, osteogenic and adipogenic differentiated hMSCs. SDC-1 KD cultures were examined for osteogenic and adipogenic lineage markers along with changes in HSPG profile and common signalling pathways implicated in hMSC lineage fate. Undifferentiated hMSC SDC-1 KD cultures exhibited a pro-adipogenic phenotype with subsequent osteogenic differentiation demonstrating enhanced maturation of osteoblasts. In cultures where SDC-1 KD was performed following initiation of differentiation, increased adipogenic gene and protein marker expression along with increased Oil Red O staining identified enhanced adipogenesis, with impaired osteogenesis also observed in these cultures. These findings implicate SDC-1 as a facilitator of the hMSC osteo-adipogenic balance during early induction of lineage differentiation.

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Suppression of Mesenchymal Stromal Cell (MSC) Differentiation in Multiple Myeloma (MM) Is Restricted to the Osteoblast Lineage

Blood ◽

10.1182/blood.v112.11.2752.2752 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2752-2752

Author(s):

Flavi a Esteve ◽

Chang Sook Hong ◽

Alissa Huston ◽

Veronica Garcia Palacios ◽

Judy Anderson ◽

...

Keyword(s):

Bone Disease ◽

Osteoblast Differentiation ◽

Bone Lesions ◽

Osteoblast Activity ◽

Lytic Bone Lesions ◽

Oil Red O Staining ◽

Osteoblast Lineage ◽

Oil Red O ◽

Msc Differentiation ◽

Group 1

Abstract Lytic bone lesions in patients with MM rarely heal even when patients are in complete remission for long periods of time. The mechanism behind this prolonged suppression of osteoblast activity is yet to be elucidated, and when or whether the osteoblast suppression can be reversed is unknown. Several possibilities could explain osteoblast suppression in MM: quantitative MSCs depletion induced by MM cells; a permanent block in MSC differentiation to the osteoblast lineage; and/or an inability of MSCs to differentiate in a hostile microenvironment. To distinguish among these possibilities, we developed a murine model of MM bone disease that permits us to assess the time course for development of osteoblast inhibition of MSC differentiation of osteoblasts, if this inhibition is reversible anytime during the process, and if the differentiation is restricted to permanent blockade of osteoblast differentiation. We generated a murine MM cell line genetically modified to contain the TK gene, which allows ablation of the cells by ganciclovir while sparing hematopoietic and stromal cell progenitors. MM cells also contained GFP for tumor estimation and a blasticidin resistance cassette for selection of transfected cells. Hematopoietic precursors and MSCs were cocultured with the MM cells in media containing ganciclovir to assess for toxic effects of this drug on cell differentiation (bystander effect). Murine MM cells or saline was also injected into the right tibia of SCID mice on day 0 and tumor lesions were documented by weekly imaging of right tibias by micro-QCT and by measurement of IgG2b in serum. Mice were treated with intraperitoneal ganciclovir or saline for 14 days starting at different time points (group 1 = 1d; group 2 = 8d; or group 3 = 14d) after tumor injection, and were sacrificed at week 5. MSCs were recovered from right tibias, cultured in osteogenic media, and alkaline phosphatase levels determined after 10 days of culture to assess osteoblast activity. MSCs were also cultured in adipogenic media, and the presence of mature adipocytes was visualized by Oil Red O staining. There was no toxic effect of ganciclovir on hematopoietic colony formation or osteoblast differentiation. Lytic bone lesions were documented in mice injected with MM cells by micro-QCT at 4 weeks in groups 2 and 3 and progressed thereafter, but not in group 1 after intratibial injection of MM cells. MSCs from group 1 mice showed greater osteoblast activity when sacrificed at 5 weeks compared to other groups. Mice in group 1 surviving 5 weeks eventually developed MM bone disease and succumbed to it at a much later time than the other groups (p<.01). Significant elevations of serum IgG2b levels were detectable at week 4 in mice from groups 2 and 3 and correlated with the development of bone lytic lesions. Harvesting cells from tumor bearing tibias yielded similar numbers of MSCs in all groups. Comparable levels of adipocytic differentiation by Oil Red O staining were observed among MSC from all groups of mice. These results demonstrate that MSC depletion cannot explain the absent osteoblast activity in this model of MM. MSC differentiation appears to be selectively blocked from the osteoblast lineage. Suppression of osteoblast activity required >24hr exposure to MM cells in vivo and correlated with relative tumor burden. Studies are underway to determine if osteoblast suppression is permanent or can be reversed in this model. This model of MM bone disease should permit the further elucidation of the mechanisms responsible for osteoblast suppression in MM, and testing of anabolic agents in a model that does not require treatment of mice with agents to eradicate MM cells and that are toxic to the marrow microenvironment.

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Therapeutic effect of Prdx1 on NAFLD mice may be related to the activation of Nrf-2/HO-1 pathway

Current Pharmaceutical Design ◽

10.2174/1381612826666201026151758 ◽

2020 ◽

Vol 26 ◽

Author(s):

Ru-Xue Bai ◽

Ying-Ying Xu ◽

Yan-Ming Chen ◽

Geng Qin ◽

Hui-Fen Wang ◽

...

Keyword(s):

Positive Staining ◽

Wild Type ◽

Mice Model ◽

Alcoholic Fatty Liver ◽

Liver Histopathology ◽

Final Weight ◽

Oil Red O Staining ◽

Alcoholic Fatty Liver Disease ◽

Oil Red O ◽

Liver Tissues

Objective: To investigate the effect of peroxiredoxin1 (Prdx1) on the methionine-choline deficient (MCD)- induced mice model of non-alcoholic fatty liver disease (NAFLD). Methods: Wild type (WT), transgenic Prdx1 over-expressing (TG) and Prdx1 knockout (KO) mice were fed with MCD diet to construct NAFLD model. General parameters was determined followed by detection with HE staining, oil red O staining, Immunofluorescence, Immunohistochemistry, qRT-PCR and Western blotting. The activities of MDA, GPX and SOD were also quantified. Results: Compared with WT + MCD group, mice in KO + MCD group showed the decresed final weight, food intake and the levels of glucose, insulin, total cholesterol and triglyceride, accompanying with the increased FFA, ALT and AST, as well as the aggravated liver histopathology, which was alleviated in TG + MCD group. Also, mice from KO + MCD group had increased F4/80 and CD68 positive staining with the upregulation of pro-inflammatory and fibrogenic factors in liver tissues than those from WT + MCD group, as well as the enhanced MDA and the reduced GPX and SOD, while TG + MCD group demonstrated improvements than the WT + MCD group. Nrf-2/HO-1 pathway in liver tissues from NFALD mice was inhibited, and Prdx1-/- can further reduce the expression of Nrf-2 and HO-1, while Prdx1 overexpression increased Nrf-2 and HO-1 expression. Conclusion: Prdx1 improved oxidative stress, inflammation and fibrosis in liver of NAFLD mice, which may be associate with the activation of Nrf-2/HO-1 pathway.

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Study on the Mechanism of Shugan Xiaozhi Fang on Cells with Non-alcoholic Fatty Liver Disease

Open Chemistry ◽

10.1515/chem-2019-0145 ◽

2019 ◽

Vol 17 (1) ◽

pp. 1328-1338

Author(s):

Yufeng Xing ◽

Chuantao Zhang ◽

Fenfen Zhai ◽

Tianran Zhou ◽

Xiang Cui ◽

...

Keyword(s):

Liver Disease ◽

Fatty Liver Disease ◽

Dose Group ◽

Control Group ◽

High Dose ◽

Model Group ◽

Alcoholic Fatty Liver ◽

Oil Red O Staining ◽

Alcoholic Fatty Liver Disease ◽

Oil Red O

AbstractCells with non-alcoholic fatty liver disease (NAFLD) were studied to determine the mechanism of liver deficiency via the AdipoR2-PPARa pathway. NAFLD cells were randomly divided into a normal control group, blank control group, model group, low dose group, medium dose group, and high dose group. The NAFLD models were established by incubating the cells with linoleic acid (LA) and palmitic acid (PA) (2:1) for 24 h. The test groups were incubated with different doses of Shugan Xiaozhi Fang extract. The pathological changes in cells that accumulated lipids were detected by Oil Red O staining. Malondialdehyde (MDA) and triglyceride (TG) levels were measured. The apoptosis of cells was evaluated by flow cytometry. The levels of AdipoR2, PPARa, CD36, acyl-CoA mRNA, and protein were confirmed by RT- PCR and Western blot. The results of the Oil Red O staining demonstrated that the NAFLD cell model was successfully established. Compared with the model group, the levels of TG and MDA in the groups that received low, medium, and high doses of Shugan Xiaozhi were significantly lower (P<0.01), and a dose effect was evident. In addition, the expression of AdipoR2, PPARa, CD36, acyl-CoA protein, and mRNA in the Shugan Xiaozhi-treated groups was upregulated. Furthermore, the levels of AdipoR2, PPAR, CD36, acyl-CoA protein, and mRNA in all drug treatment groups that were extracted from L-O2 normal human hepatocytes were significantly upregulated (P<0.01). Moreover, the factor pattern of HepG2 human liver carcinoma cells was similar to that of L-O2. The levels of AdipoR, CD36, acyl-CoA, and AdipoR mRNA in the HepG2 low group were increased (P<0.05). AdipoR, PPAR, CD36, and acyl-CoA protein levels and AdipoR mRNA expression were significantly increased in the intermediate dose group and high dose group (P<0.01). Shugan Xiaozhi Fang attenuates hepatic lipid deposition in NAFLD induced by incubating with LA and PA for 24 h, which is associated with the activation of the AdipoR2-PPARα pathway.

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The Effects of Andrographolide on the Enhancement of Chondrogenesis and Osteogenesis in Human Suprapatellar Fat Pad Derived Mesenchymal Stem Cells

Molecules ◽

10.3390/molecules26071831 ◽

2021 ◽

Vol 26 (7) ◽

pp. 1831

Author(s):

Thitianan Kulsirirat ◽

Sittisak Honsawek ◽

Mariko Takeda-Morishita ◽

Nuttanan Sinchaipanid ◽

Wanvisa Udomsinprasert ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Adipogenic Differentiation ◽

Human Mesenchymal Stem Cells ◽

Fat Pad ◽

Alizarin Red ◽

Lineage Differentiation ◽

Expression Of Genes ◽

Dose Dependent ◽

Oil Red O Staining

Andrographolide is a labdane diterpenoid herb, which is isolated from the leaves of Andrographis paniculata, and widely used for its potential medical properties. However, there are no reports on the effects of andrographolide on the human suprapatellar fat pad of osteoarthritis patients. In the present study, our goal was to evaluate the innovative effects of andrographolide on viability and Tri-lineage differentiation of human mesenchymal stem cells from suprapatellar fat pad tissues. The results revealed that andrographolide had no cytotoxic effects when the concentration was less than 12.5 µM. Interestingly, andrographolide had significantly enhanced, dose dependent, osteogenesis and chondrogenesis as evidenced by a significantly intensified stain for Alizarin Red S, Toluidine Blue and Alcian Blue. Moreover, andrographolide can upregulate the expression of genes related to osteogenic and chondrogenic differentiation, including Runx2, OPN, Sox9, and Aggrecan in mesenchymal stem cells from human suprapatellar fat pad tissues. In contrast, andrographolide suppressed adipogenic differentiation as evidenced by significantly diminished Oil Red O staining and expression levels for adipogenic-specific genes for PPAR-γ2 and LPL. These findings confirm that andrographolide can specifically enhance osteogenesis and chondrogenesis of mesenchymal stem cells from human suprapatellar fat pad tissues. It has potential as a therapeutic agent derived from natural sources for regenerative medicine.

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Type III Collagen is Required for Adipogenesis and Actin Stress Fibre Formation in 3T3-L1 Preadipocytes

Biomolecules ◽

10.3390/biom11020156 ◽

2021 ◽

Vol 11 (2) ◽

pp. 156

Author(s):

Mohammad Al Hasan ◽

Patricia E. Martin ◽

Xinhua Shu ◽

Steven Patterson ◽

Chris Bartholomew

Keyword(s):

Quantitative Polymerase Chain Reaction ◽

Type Iii Collagen ◽

Type Iii ◽

Actin Stress Fibre ◽

Matrix Gene ◽

Gene Transcripts ◽

Polymerase Chain ◽

Oil Red O Staining ◽

Oil Red O

GPR56 is required for the adipogenesis of preadipocytes, and the role of one of its ligands, type III collagen (ColIII), was investigated here. ColIII expression was examined by reverse transcription quantitative polymerase chain reaction, immunoblotting and immunostaining, and its function investigated by knockdown and genome editing in 3T3-L1 cells. Adipogenesis was assessed by oil red O staining of neutral cell lipids and production of established marker and regulator proteins. siRNA-mediated knockdown significantly reduced Col3a1 transcripts, ColIII protein and lipid accumulation in 3T3-L1 differentiating cells. Col3a1−/− 3T3-L1 genome-edited cell lines abolished adipogenesis, demonstrated by a dramatic reduction in adipogenic moderators: Pparγ2 (88%) and C/ebpα (96%) as well as markers aP2 (93%) and oil red O staining (80%). Col3a1−/− 3T3-L1 cells displayed reduced cell adhesion, sustained active β-catenin and deregulation of fibronectin (Fn) and collagen (Col4a1, Col6a1) extracellular matrix gene transcripts. Col3a1−/− 3T3-L1 cells also had dramatically reduced actin stress fibres. We conclude that ColIII is required for 3T3-L1 preadipocyte adipogenesis as well as the formation of actin stress fibres. The phenotype of Col3a1−/− 3T3-L1 cells is very similar to that of Gpr56−/− 3T3-L1 cells, suggesting a functional relationship between ColIII and Gpr56 in preadipocytes.

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Genetic characteristics of Bursaphelenchus xylophilus third-stage dispersal juveniles

Scientific Reports ◽

10.1038/s41598-021-82343-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Qiaoli Chen ◽

Ruizhi Zhang ◽

Danlei Li ◽

Feng Wang

Keyword(s):

Fat Metabolism ◽

Bursaphelenchus Xylophilus ◽

Pinewood Nematode ◽

Genetic Characteristics ◽

Highest Weight ◽

Homologous Genes ◽

Third Stage ◽

Highly Correlated ◽

Oil Red O Staining ◽

Oil Red O

AbstractThe third-stage dispersal juvenile (DJ3) of pinewood nematode (PWN) is highly associated with low-temperature survival and spread of the nematode. Oil-Red-O staining showed that its lipid content was significantly higher compared with other PWN stages. Weighted gene coexpression network analysis identified that genes in the pink module were highly related to DJ3 induced in the laboratory (DJ3-lab). These genes were arranged according to their gene significance (GS) to DJ3-lab. Of the top 30 genes with the highest GS, seven were found to be highly homologous to the cysteine protease family cathepsin 1 (CATH1). The top 30 genes with the highest weight value to each of the seven genes in the pink module were selected, and finally 35 genes were obtained. Between these seven CATH1 homologous genes and their 35 highly related genes, 15 were related to fat metabolism or autophagy. These autophagy-related genes were also found to be highly correlated with other genes in the pink module, suggesting that autophagy might be involved in the mechanism of longevity in DJ3 and the formation of DJ3 by regulating genes related to fat metabolism.

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Pathogenic Microenvironment from Diabetic–Obese Visceral and Subcutaneous Adipocytes Activating Differentiation of Human Healthy Preadipocytes Increases Intracellular Fat, Effect of the Apocarotenoid Crocetin

Nutrients ◽

10.3390/nu13031032 ◽

2021 ◽

Vol 13 (3) ◽

pp. 1032

Author(s):

Lesgui Alviz ◽

David Tebar-García ◽

Raquel Lopez-Rosa ◽

Eva M. Galan-Moya ◽

Natalia Moratalla-López ◽

...

Keyword(s):

Adipose Tissue ◽

Subcutaneous Adipose Tissue ◽

Bioactive Components ◽

Hypertrophic Growth ◽

Visceral Adipose ◽

Subcutaneous Adipose ◽

Oil Red O Staining ◽

Oil Red O ◽

Excessive Accumulation

In diabetes mellitus type 2 (DM2), developed obesity is referred to as diabesity. Implementation of a healthy diet, such as the Mediterranean, prevents diabesity. Saffron is frequently used in this diet because of its bioactive components, such as crocetin (CCT), exhibit healthful properties. It is well known that obesity, defined as an excessive accumulation of fat, leads to cardiometabolic pathology through adiposopathy or hypertrophic growth of adipose tissue (AT).This is related to an impaired adipogenic process or death of adipocytes by obesogenic signals. We aimed to evaluate the effect of the pathogenic microenvironment and CCT, activating differentiation of healthy preadipocytes (PA). For this, we used human cryopreserved PA from visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) depots obtained from healthy and obese-DM2 donors. We studied the effect of a metabolically detrimental (diabesogenic) environment, generated by obese-DM2 adipocytes from VAT (VdDM) or SAT (SdDM), on the viability and accumulation of intracellular fat of adipocytes differentiated from healthy PA, in the presence or absence of CCT (1 or 10 μM). Intracellular fat was quantified by Oil Red O staining. Cytotoxicity was measured using the MTT assay. Our results showed that diabesogenic conditions induce cytotoxicity and provide a proadipogenic environment only for visceral PA. CCT at 10 μM acted as an antiadipogenic and cytoprotective compound.

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