Orexin-A Exerts Equivocal Role in Atherosclerosis Process Depending on the Duration of Exposure: In Vitro Study

Orexin-A is a peptide hormone that plays a crucial role in feeding regulation and energy homeostasis. Diurnal intermittent fasting (DIF) has been found to increase orexin-A plasma levels during fasting hours, while Ramadan fasting which resembles DIF, has led to beneficial effects on endothelial function. Herein, we aimed to investigate the effects of orexin-A on the expression of molecules involved in the atherogenesis process: Monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) and tissue inhibitor of metalloproteinase-1 and 2 (TIMP-1 and TIMP-2), in human aortic endothelial cells (HAECs). HAECs were incubated with orexin-A at concentrations of 40 ng/mL, 200 ng/mL and 400 ng/mL for 6, 12 and 24 h. The mRNA levels of MCP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 and orexin-1 receptor were measured by real-time qPCR. We also evaluated the MMP-2, p38, phospho-p38, NF-κΒ/p65 as well as TIMP-1 protein levels by Western blot and ELISA, respectively. MMP-2 activity was measured by gelatin zymography. Short-term 6-h incubation of HAECs with orexin-A at a high concentration (400 ng/mL) decreased MCP-1, MMP-2 expression, MMP-2/TIMP-1 ratio (p < 0.05), and MMP-2 activity, while incubation for 24 h increased MCP-1, MMP-2 expression (p < 0.05), MMP-2/TIMP-1 and MMP-2/TIMP-2 ratio (p < 0.01 and p < 0.05, respectively) as well as MMP-2 activity. The dual effects of orexin-A are mediated, at least in part, via regulation of p38 and NF-κΒ pathway. Orexin-A may have an equivocal role in atherosclerosis process with its effects depending on the duration of exposure.

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Similar Features, Different Behaviors: A Comparative In Vitro Study of the Adipogenic Potential of Stem Cells from Human Follicle, Dental Pulp, and Periodontal Ligament

Journal of Personalized Medicine ◽

10.3390/jpm11080738 ◽

2021 ◽

Vol 11 (8) ◽

pp. 738

Author(s):

Melissa D. Mercado-Rubio ◽

Erick Pérez-Argueta ◽

Alejandro Zepeda-Pedreguera ◽

Fernando J. Aguilar-Ayala ◽

Ricardo Peñaloza-Cuevas ◽

...

Keyword(s):

Stem Cells ◽

Dental Pulp ◽

Periodontal Ligament ◽

Adipogenic Differentiation ◽

In Vitro Study ◽

Mrna Levels ◽

Dental Tissue ◽

Periodontal Ligament Stem Cells ◽

Differentiation Potential

Dental tissue-derived mesenchymal stem cells (DT-MSCs) are a promising resource for tissue regeneration due to their multilineage potential. Despite accumulating data regarding the biology and differentiation potential of DT-MSCs, few studies have investigated their adipogenic capacity. In this study, we have investigated the mesenchymal features of dental pulp stem cells (DPSCs), as well as the in vitro effects of different adipogenic media on these cells, and compared them to those of periodontal ligament stem cells (PLSCs) and dental follicle stem cells (DFSCs). DFSC, PLSCs, and DPSCs exhibit similar morphology and proliferation capacity, but they differ in their self-renewal ability and expression of stemness markers (e.g OCT4 and c-MYC). Interestingly, DFSCs and PLSCs exhibited more lipid accumulation than DPSCs when induced to adipogenic differentiation. In addition, the mRNA levels of adipogenic markers (PPAR, LPL, and ADIPOQ) were significantly higher in DFSCs and PLSCs than in DPSCs, which could be related to the differences in the adipogenic commitment in those cells. These findings reveal that the adipogenic capacity differ among DT-MSCs, features that might be advantageous to increasing our understanding about the developmental origins and regulation of adipogenic commitment.

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Cellular effects of glycine and trehalose air-polishing powders on human gingival fibroblasts in vitro

Clinical Oral Investigations ◽

10.1007/s00784-021-04130-0 ◽

2021 ◽

Author(s):

Jens Weusmann ◽

James Deschner ◽

Jean-Claude Imber ◽

Anna Damanaki ◽

Natalia D. P. Leguizamón ◽

...

Keyword(s):

Wound Healing ◽

Cell Function ◽

Nuclear Translocation ◽

In Vitro Study ◽

Human Gingival Fibroblasts ◽

Mrna Levels ◽

Gingival Fibroblasts ◽

Air Polishing ◽

Wide Range

Abstract Objectives Air-polishing has been used in the treatment of periodontitis and gingivitis for years. The introduction of low-abrasive powders has enabled the use of air-polishing devices for subgingival therapy. Within the last decade, a wide range of different low-abrasive powders for subgingival use has been established. In this study, the effects of a glycine powder and a trehalose powder on human gingival fibroblasts (HGF) were investigated. Methods HGF were derived from three systemically and periodontally healthy donors. After 24 h and 48 h of incubation time, mRNA levels, and after 48 h, protein levels of TNFα, IL-8, CCL2, and VEGF were determined. In addition, NF-κB p65 nuclear translocation and in vitro wound healing were assessed. Statistical analysis was performed by ANOVA and post hoc Dunnett’s and Tukey’s tests (p < 0.05). Results Glycine powder significantly increased the expression of proinflammatory genes and showed exploitation of the NF-κB pathway, albeit trehalose powder hardly interfered with cell function and did not trigger the NF-κB pathway. In contrast to trehalose, glycine showed a significant inhibitory effect on the in vitro wound healing rate. Conclusion Subgingivally applicable powders for air-polishing devices can regulate cell viability and proliferation as well as cytokine expression. Our in vitro study suggests that the above powders may influence HGF via direct cell effects. Trehalose appears to be relatively inert compared to glycine powder.

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Leakage of Endotoxins through the Endotoxin Retentive Filter: An in vitro Study

Blood Purification ◽

10.1159/000520792 ◽

2022 ◽

pp. 1-9

Author(s):

Hiroshi Nozaki ◽

Yoshihiro Tange ◽

Yoji Inada ◽

Takashi Uchino ◽

Nakanobu Azuma

Keyword(s):

Sodium Hypochlorite ◽

Peracetic Acid ◽

In Vitro Study ◽

Dialysis Fluid ◽

High Concentration ◽

Membrane Pores ◽

Polyether Sulfone ◽

Repeated Use ◽

High Concentrations

Introduction: Ultrapurification of dialysis fluid has enabled highly efficient dialysis treatments. Online hemodiafiltration is one such treatment that uses a purified dialysis fluid as a supplemental fluid. In this method, an endotoxin retentive filter (ETRF) is used in the final step of dialysis fluid purification, with the aim of preventing leakage of endotoxins. Sodium hypochlorite and peracetic acid are used as disinfecting agents for the dialysis fluid pipes containing the ETRF; however, the effects of these agents on ETRF membrane pores have not been fully clarified. Methods: Water permeability (flux) and endotoxin permeability were assessed in 3 types of ETRFs made with different membrane materials: polyester polymer alloy (PEPA), polyether sulfone (PES), and polysulfone (PS). High-concentration sodium hypochlorite and 2 types of peracetic acid were used as disinfecting agents, and the changes in flux and the endotoxin sieving coefficient (SC) were measured. Results: After repeated use of high concentrations of sodium hypochlorite and peracetic acid, the PEPA and PES ETRFs did not permit passage of endotoxins, regardless of their flux. However, in the PS ETRF, the flux and endotoxin SC increased with the number of cleaning cycles. No differences were observed according to the concentration of peracetic acid disinfecting agents. Conclusion: PEPA and PES ETRFs completely prevent endotoxin leakage and can be disinfected at concentrations higher than the conventionally recommended concentration without affecting pore expansion. Even new PS ETRFs have low levels of endotoxin leakage, which increase after disinfection cycles using sodium hypochlorite and peracetic acid.

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Evaluation of Sealing Materials Adhesion to Enamel An in vitro study

Materiale Plastice ◽

10.37358/mp.17.1.4801 ◽

2017 ◽

Vol 54 (1) ◽

pp. 129-132

Author(s):

Andreea Simona Pop ◽

Radu Septimiu Campian ◽

Mariana Pacurar ◽

Elina Teodorescu ◽

Olimpia Bunta ◽

...

Keyword(s):

In Vitro Study ◽

Specific Method ◽

High Concentration ◽

Posterior Teeth ◽

Inorganic Particles ◽

Material Homogeneity ◽

Materials Used ◽

High Degree ◽

Pits And Fissures

Sealing the pits and fissures of posterior teeth represents a local and specific method of caries prevention. The aim of this study was to evaluate the adhesion of two materials used in sealing pits and fissures: Pitt and Fisure and Fissurit FX (Voco), with the help of the scanning electronic microscope (SEM). The results of the study revealed a much higher quality of the Fissurit FX (Voco) product both in terms of adhesion to the enamel and material homogeneity. The Pitt and Fisure product showed a high degree of detachment from the enamel, marginal infiltration, large particles and a high concentration of inorganic particles.

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Bovine granulosa cells express extracellular matrix proteins and their regulators during luteinization in culture

Reproduction Fertility and Development ◽

10.1071/rd9960259 ◽

1996 ◽

Vol 8 (2) ◽

pp. 259 ◽

Cited By ~ 15

Author(s):

Y Zhao ◽

MR Luck

Keyword(s):

Extracellular Matrix ◽

Granulosa Cells ◽

Culture Media ◽

Gelatin Zymography ◽

Fold Increase ◽

Tissue Inhibitor Of Metalloproteinase ◽

Subunit Gene ◽

Gelatinase Activity ◽

Cell Extracts

This study investigated the ability of bovine granulosa cells to express and secrete collagen, metalloproteinase (MMP) activity and a tissue inhibitor of metalloproteinase (TIMP-1) during luteinization in vitro. Cells from mature (1-2 mL fluid volume) bovine follicles were cultured over 4 days in serum-free medium. Their luteinization during culture was confirmed by a 10-fold increase in progesterone secretion. Samples of cell extracts, culture media and follicular fluid were subjected to Western blotting to identify secreted proteins and to gelatin zymography to detect enzyme activity. Poly A+ RNA, isolated from cells before and after culture, was probed to detect expression of collagen alpha 1(I), collagen alpha 3(IV) and TIMP-1. The results revealed that: (1) the collagen alpha 1(I) subunit gene was expressed in cells before culture but with greater intensity by Day 4 culture; collagen I protein, on the other hand, was not detectable in culture medium; (2) the collagen alpha 3(IV) subunit gene was expressed at a low level in uncultured cells and could be detected on Day 4 of culture; low amounts of the protein were detected in medium; (3) a 92-kDa band of gelatinase activity (presumed MMP-9) was present in all medium samples, together with bands of unidentified activity; and (5) the TIMP-1 gene was expressed in uncultured cells but its expression increased markedly up to Day 4 of culture. These results show that granulosa luteinization is associated with an increase in the expression of collagen, collagen-degrading enzymes and TIMP-1. Collagen protein, however, may be only poorly synthesized in this culture model. The results suggest that granulosa-derived cells are a likely source of components of the extracellular matrix during post-ovulatory remodelling of early luteal tissue.

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C–C motif chemokine receptor 2 as a novel intermediate in the ovulatory cascade

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gaaa020 ◽

2020 ◽

Vol 26 (5) ◽

pp. 289-300

Author(s):

JP Jaworski ◽

M Urrutia ◽

E Dascal ◽

G Jaita ◽

MC Peluffo

Keyword(s):

Oocyte Maturation ◽

Chemokine Receptor ◽

Follicle Cells ◽

Mrna Levels ◽

Monocyte Chemoattractant Protein 1 ◽

Monocyte Chemoattractant ◽

Monocyte Chemoattractant Protein ◽

Cumulus Oocyte Complex ◽

Key Genes

Abstract Expression of immune function genes within follicle cells has been reported in ovaries from many species. Recent work from our laboratory showed a direct effect of the monocyte chemoattractant protein 1/C-C motif chemokine receptor 2 system within the feline cumulus oocyte complex, by increasing the mRNA levels of key genes involved in the ovulatory cascade in vitro. Studies were designed to evaluate if C–C motif chemokine receptor 2 acts as a novel mediator of the ovulatory cascade in vitro. Therefore, feline cumulus oocyte complexes were cultured in the presence or absence of a highly selective C–C motif chemokine receptor 2 antagonist together with known inducers of cumulus–oocyte expansion and/or oocyte maturation to assess mRNA expression of key genes related to periovulatory events in other species as well as oocyte maturation. Also, the effects of recombinant monocyte chemoattractant protein 1 on spontaneous or gonadotrophin-induced oocyte maturation were assessed. This is an in vitro system using isolated cumulus oocyte complexes from feline ovaries. The present study reveals the modulation of several key ovulatory genes by a highly selective C–C motif chemokine receptor 2 antagonist. However, this antagonist was not enough to block the oocyte maturation induced by gonadotropins or amphiregulin. Nonetheless, recombinant monocyte chemoattractant protein 1 had a significant effect on spontaneous oocyte maturation, increasing the percentage of metaphase II stage oocytes in comparison to the control. This is the first study in any species to establish C–C motif chemokine receptor 2 as a mediator of some actions of the mid-cycle gonadotrophin surge.

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A preprogalanin cDNA from the turtle pituitary and regulation of its gene expression

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00452.2006 ◽

2007 ◽

Vol 292 (4) ◽

pp. R1649-R1656 ◽

Cited By ~ 2

Author(s):

John Yuh-Lin Yu ◽

Chin-Hon Pon ◽

Hui-Chen Ku ◽

Chih-Ting Wang ◽

Yung-Hsi Kao

Keyword(s):

Mrna Expression ◽

Untranslated Region ◽

Signal Sequence ◽

Biological Effects ◽

In Vitro Study ◽

The Body ◽

Mrna Levels ◽

Coding Region ◽

Reading Frame

Galanin is a hormone 29 or 30 amino acids (aa) long that is widely distributed within the body and exerts numerous biological effects in vertebrates. To fully understand its physiological roles in reptiles, we analyzed preprogalanin cDNA structure and expression in the turtle pituitary. Using the Chinese soft-shell turtle ( Pelodiscus sinensis order Testudines), we obtained a 672-base pair (bp) cDNA containing a 99-bp 5′-untranslated region, a 324-bp preprogalanin coding region, and a 249-bp 3′-untranslated region. The open-reading frame encoded a 108-aa preprogalanin protein with a putative 23-aa signal sequence at the NH2 terminus. Based on the location of putative Lys-Arg dibasic cleavage sites and an amidation signal of Gly-Lys-Arg, we propose that turtle preprogalanin is processed to yield a 29-aa galanin peptide with Gly1 and Thr29 substitutions and a COOH-terminal amidation. Sequence comparison revealed that turtle preprogalanin and galanin-29 had 48–81% and 76–96% aa identities with those of other vertebrates, respectively, suggesting their conservative nature. Expression of the turtle galanin gene was detected in the pituitary, brain, hypothalamus, stomach, liver, pancreas, testes, ovaries, and intestines, but not in the adipose or muscle tissues, suggesting tissue-dependent differences. An in vitro study that used pituitary tissue culture indicated that treatment with 17β-estradiol, testosterone, or gonadotropin-releasing hormone resulted in increased galanin mRNA expression with dose- or time-dependent differences, whereas leptin and neuropeptide Y reduced galanin mRNA levels. These results suggest a hormone-dependent effect on hypophyseal galanin mRNA expression.

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Repression of Matrix Metalloproteinases and Cytokine Secretion in Glioblastoma by Targeting K+ Channel: An in Vitro Study

Basic and Clinical Neuroscience Journal ◽

10.32598/bcn.2021.1693.1 ◽

2021 ◽

Author(s):

Farshid Saadat ◽

◽

Zohreh Zareighane ◽

Farnaz Safavifar ◽

Seyedeh Zohreh Jalali ◽

...

Keyword(s):

Matrix Metalloproteinases ◽

Cell Viability ◽

Malignant Tumors ◽

Cell Model ◽

Gelatin Zymography ◽

In Vitro Study ◽

K Channel ◽

Kv Channels ◽

Vitro Model

Introduction: Glioblastoma is an aggressive malignancy of human brain with poorly understood pathogenesis. Voltage-gated potassium (Kv) channels and Matrix metalloproteinases (MMPs) are highly expressed in malignant tumors and involved in the progression and metastasis of glioblastoma. The purpose of this study was to determine whether a voltage-dependent potassium channel blocker could modulate astrocytes as a cell which involved in immunopathogenesis of glioblastoma. Methods: The cytotoxic effect of 4-aminopyridine (4-AP) at different doses in cell model of glioblastoma was measured by MTT assay. ELISA technique and gelatin zymography were used to assess cytokines levels and MMP-9 after 4-AP treatment, respectively. Results: Cytotoxicity analysis showed that cell viability reduced by increasing 4-AP level and cell growth reduced gradually by removing 4-AP from cell medium. 4-AP inhibits secretion of IL-6 and IL-1 (p<0.05). MMP9 activity significantly inhibits with increased 4-AP dose as compared to non-treated cells. Conclusion: Reduction of cell viability, IL-6 secretion and MMP-9 activity in an in vitro model of glioblastoma, might be assumed 4-AP as an agent for chemoprevention of cancer.

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The selective NLRP3 inhibitor MCC950 hinders atherosclerosis development by attenuating inflammation and pyroptosis in macrophages

Scientific Reports ◽

10.1038/s41598-021-98437-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Wenyun Zeng ◽

Danbin Wu ◽

Yingxin Sun ◽

Yanrong Suo ◽

Qun Yu ◽

...

Keyword(s):

Nlrp3 Inflammasome ◽

High Fat Diet ◽

In Vitro Study ◽

Inflammasome Activation ◽

Mrna Levels ◽

High Fat ◽

Nlrp3 Inflammasome Activation ◽

Atherosclerosis Development

AbstractNLRP3 inflammasome is a vital player in macrophages pyroptosis, which is a type of proinflammatory cell-death and takes part in the pathogenesis of atherosclerosis. In this study, we used apoE−/− mice and ox-LDL induced THP-1 derived macrophages to explore the mechanisms of MCC950, a selective NLRP3 inhibitor in treating atherosclerosis. For the in vivo study, MCC950 was intraperitoneal injected to 8-week-old apoE−/− mice fed with high-fat diet for 12 weeks. For the in vitro study, THP-1 derived macrophages were treated with ox-LDL and MCC950 for 48 h. MCC950 administration reduced plaque areas and macrophages contents, but did not improve the serum lipid profiles in aortic root of apoE−/− mice. MCC950 inhibited the activation of NLRP3/ASC/Caspase-1/GSDMD-N axis, and alleviated macrophages pyroptosis and the production of IL-1β and IL-18 both in aorta and in cell lysates. However, MCC950 did not affect the expression of TLR4 or the mRNA levels of NLRP3 inflammasome and its downstream proteins, suggesting that MCC950 had no effects on the priming of NLRP3 inflammasome activation in macrophages. The anti-atherosclerotic mechanisms of MCC950 on attenuating macrophages inflammation and pyroptosis involved in inhibiting the assembly and activation of NLRP3 inflammasome, rather than interrupting its priming.

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TIMP-2 secreted by monocytes is a potent suppressor of invadopodia formation in pancreatic cancer cells

10.21203/rs.2.10555/v1 ◽

2019 ◽

Author(s):

Christian Benzing ◽

Hoyin Lam ◽

Chi Man Tsang ◽

Yoana Arroyo-Berdugo ◽

Yolanda Calle-Patino ◽

...

Keyword(s):

Primary Tumour ◽

In Vitro Study ◽

Tumour Microenvironment ◽

Tissue Inhibitor Of Metalloproteinase ◽

Ductal Adenocarcinoma ◽

Pancreatic Cancer Cells ◽

Therapeutic Opportunity ◽

The Impact ◽

Invadopodia Formation

Abstract Background Monocytes are a major component of the tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC). However, the complex interactions between tumor cells and monocytes and their role in tumor invasion have not been fully established. Methods In this in vitro study, to specifically test the impact of interaction on invasive potential, two PDAC cell lines PaTu8902 and CFPAC-1 were selected on their ability to form invasive adhesions, otherwise known as invadopodia. Co-culture experiments were performed using undifferentiated THP1 monocytes. Results When the PDAC cells were co-cultured with undifferentiated THP1 monocytes invadopodia formation was significantly suppressed. Moreover, conditioned media of THP1 cells (CM) was also able to suppress invadopodia formation. Further investigation revealed that both tissue inhibitor of metalloproteinase (TIMP) 1 and 2 were present in the CM. However, suppression of invadopodia formation was found that was specific to TIMP2 activity. Conclusions Our findings indicate that TIMP2 levels in the tumour microenvironment may have prognostic value in patients with PDAC. Furthermore, activation of TIMP2 expressing monocytes in the primary tumour could present a potential therapeutic opportunity to suppress cell invasion in PDAC.

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