Reduced TGF-β Expression and CD206-Positive Resident Macrophages in the Intervertebral Discs of Aged Mice

Age is a key factor in intervertebral disc (IVD) degeneration; however, the changes that occur in IVDs with age are not fully understood. Tissue-resident macrophages are critical for tissue homeostasis and are regulated by transforming growth factor- (TGF-) β. We examined changes in the proportion of resident macrophages in young versus aged mice and the role of TGF-β in regulating resident macrophages in IVDs. IVDs were harvested from 4-month (young) and 18-month-old (aged) C57BL/6J mice. The proportion of macrophages in IVDs was determined using flow cytometry ( n = 5 for each time point) and the expression of Cd11b, Cd206, and Tgfb genes, which encode CD11b, CD206, and TGF-β protein, respectively, using real-time PCR. To study the role of TGF-β in the polarization of resident macrophages, resident macrophages isolated from IVDs from young and aged mice were treated with recombinant TGF-β with and without a TGF-β inhibitor (SB431542). Additionally, SB431542 was intraperitoneally injected into young and aged mice, and Cd206 expression was examined using real-time PCR ( n = 10 for each time point). The proportion of CD11b+ and CD11b+ CD206+ cells was significantly reduced in aged versus young mice, as was Cd11b, Cd206, and Tgfb expression. TGF-β/IL10 stimulation significantly increased the expression of Cd206, an M2 macrophage marker, in disc macrophages from both young and aged mice. Meanwhile, administration of a TGF-β inhibitor significantly reduced Cd206 expression compared to vehicle control in both groups. Conclusion. Resident macrophages decrease with age in IVDs, which may be associated with the concomitant decrease in TGF-β. Our findings provide new insight into the mechanisms of age-related IVD pathology.

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Abstract 65: Restoration of Growth Differentiation Factor 11 Protects Against Stroke in Mice

Stroke ◽

10.1161/str.47.suppl_1.65 ◽

2016 ◽

Vol 47 (suppl_1) ◽

Author(s):

Anjali Chauhan ◽

Jacob Hudobenko ◽

Anthony Patrizz ◽

Louise D McCullough

Keyword(s):

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

The Elderly ◽

Vehicle Group ◽

Peripheral Tissues ◽

Delayed Treatment ◽

Aged Mice ◽

Infarct Area ◽

Age Related

Introduction: GDF 11 is a member of the transforming growth factor β superfamily. Loss of GDF11 occurs with aging and declining levels correlate with several detrimental age-associated phenotypes in both peripheral tissues and brain. Restoration of GDF11 enhances neurogenesis and cognitive function in aged mice. Brain expression of GDF11 has not been investigated after stroke. Stroke differentially affects the elderly. In this work we examined the role of GDF11 in aging, stroke and its potential utility as a neuroprotective agent. Methods: Male C57/BL6NCrl young (2-3 months) and aged (19-21) mice were used. Brain GDF11 expression was evaluated in young and aged mice by western blot. Focal ischemia was induced with a transient middle cerebral artery occlusion (MCAO). Mice were randomly assigned into two groups and were subjected to 90 min MCAO. Group 1 received vehicle (phosphate buffered saline) and group 2 was administered rGDF11 (100 ug/kg., ip) at the onset of ischemia. In additional experiments, the efficacy of delayed treatment (3 h after ischemia) with rGDF11 was tested. These mice were subjected to a 60 min MCAO. Mice were euthanized after 24 hours and 7 days respectively and brains were harvested to estimate infarct area. Results: A significant decrease in brain GDF11 levels was observed in aged mice as compared to young (p<0.05). Additionally, a significant decline in brain GDF11 expression was observed after stroke at 24 hours vs. sham groups (p<0.05). A significant decrease in cortical and hemispheric infarct area was observed in the rGDF11 group (cortical 48.73±1.05; hemisphere 49.68±3.58) as compared to vehicle group (60.54±4.88; 61.35±6.03), when GDF was administered at the time of ischemia. Delayed treatment with rGDF11 also reduced infarct at 7 days. Conclusions: Brain GDF11 levels decline with age and after stroke. Supplementation with rGDF11 ameliorates stroke induced injury in young mice at 24h and 7 days. These finding suggest potential role of GDF11 in age and stroke. Restoration of age-related loss of GDF may be a viable therapy for stroke.

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MicroRNA-296-5p inhibits cell metastasis and invasion in nasopharyngeal carcinoma by reversing transforming growth factor-β-induced epithelial–mesenchymal transition

Cellular & Molecular Biology Letters ◽

10.1186/s11658-020-00240-x ◽

2020 ◽

Vol 25 (1) ◽

Author(s):

Meihui Chen ◽

Chen Chen ◽

Haiqing Luo ◽

Jing Ren ◽

Qiuqin Dai ◽

...

Keyword(s):

Nasopharyngeal Carcinoma ◽

Real Time ◽

Real Time Pcr ◽

Transforming Growth Factor ◽

Epithelial Mesenchymal Transition ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Mesenchymal Transition ◽

Cell Metastasis

Abstract Aim To explore the effect of miR-296-5p on the metastasis of nasopharyngeal carcinoma (NPC) cells and investigate the underlying mechanism. Methods The expressions of miR-296-5p in NPC tissues and cells were determined using GSE32920 database analysis and real-time PCR and miRNA microarray assays. An miR-296-5p mimic and inhibitor were transfected into NPC cells. Then, immunofluorescence imaging, scratch wound-healing, transwell migration and invasion assays were used to observe the effects of miR-296-5p on cell metastasis and invasion. Real-time PCR and western blotting were carried out to detect the expressions of genes and proteins related to epithelial–mesenchymal transition (EMT). A dual luciferase reporter assay was used to identify whether TGF-β is the target gene of miR-296-5p. Finally, TGF-β expression plasmids were transfected into NPC cells to verify the role of TGF-β in the miR-296-5p-mediated inhibition of nasopharyngeal carcinoma cell metastasis. Results Our results show that miR-296-5p inhibits the migratory and invasive capacities of NPC cells by targeting TGF-β, which suppresses EMT. Importantly, the miR-296-5p level was significantly lower in human NPC tissues than in adjacent normal tissues. It also negatively correlated with TGF-β and was significantly associated with the lymph node metastasis of patients with NPC. Conclusions Our findings show that miR-296-5p represses the EMT-related metastasis of NPC by targeting TGF-β. This provides new insight into the role of miR-296-5p in regulating NPC metastasis and invasiveness.

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The Nutraceutical N-Palmitoylethanolamide (PEA) Reveals Widespread Molecular Effects Unmasking New Therapeutic Targets in Murine Varicocele

Nutrients ◽

10.3390/nu13030734 ◽

2021 ◽

Vol 13 (3) ◽

pp. 734

Author(s):

Pietro Antonuccio ◽

Herbert Ryan Marini ◽

Antonio Micali ◽

Carmelo Romeo ◽

Roberta Granese ◽

...

Keyword(s):

Transforming Growth Factor ◽

Therapeutic Targets ◽

Sham Operation ◽

Peroxisome Proliferator ◽

Peroxisome Proliferator Activated Receptor ◽

Pyrin Domain ◽

Age Related ◽

Extracellular Signal ◽

Morphometric Assessment

Varicocele is an age-related disease with no current medical treatments positively impacting infertility. Toll-like receptor 4 (TLR4) expression is present in normal testis with an involvement in the immunological reactions. The role of peroxisome proliferator-activated receptor-α (PPAR-α), a nuclear receptor, in fertility is still unclear. N-Palmitoylethanolamide (PEA), an emerging nutraceutical compound present in plants and animal foods, is an endogenous PPAR-α agonist with well-demonstrated anti-inflammatory and analgesics characteristics. In this model of mice varicocele, PPAR-α and TLR4 receptors’ roles were investigated through the administration of ultra-micronized PEA (PEA-um). Male wild-type (WT), PPAR-α knockout (KO), and TLR4 KO mice were used. A group underwent sham operation and administration of vehicle or PEA-um (10 mg/kg i.p.) for 21 days. Another group (WT, PPAR-α KO, and TLR4 KO) underwent surgical varicocele and was treated with vehicle or PEA-um (10 mg/kg i.p.) for 21 days. At the end of treatments, all animals were euthanized. Both operated and contralateral testes were processed for histological and morphometric assessment, for PPAR-α, TLR4, occludin, and claudin-11 immunohistochemistry and for PPAR-α, TLR4, transforming growth factor-beta3 (TGF-β3), phospho-extracellular signal-Regulated-Kinase (p-ERK) 1/2, and nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) Western blot analysis. Collectively, our data showed that administration of PEA-um revealed a key role of PPAR-α and TLR4 in varicocele pathophysiology, unmasking new nutraceutical therapeutic targets for future varicocele research and supporting surgical management of male infertility.

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Identifying the etiologic role of Parvovirus B19 in non-immune hydrops fetalis by histopathology, immunohistochemistry and nucleic acid testing: a retrospective study

Open Medicine ◽

10.2478/s11536-007-0029-z ◽

2007 ◽

Vol 2 (3) ◽

pp. 271-279 ◽

Cited By ~ 1

Author(s):

Koray Ergunay ◽

Gulcin Altinok ◽

Bora Gurel ◽

Ahmet Pinar ◽

Arzu Sungur ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

San Francisco ◽

Real Time Pcr ◽

Nested Pcr ◽

Parvovirus B19 ◽

Etiologic Agent ◽

Hydrops Fetalis ◽

Nucleic Acid Detection

AbstractIntrauterine Parvovirus B19 infections may cause fetal anemia, non-immune hydrops fetalis or abortion. This study focuses on the pathogenic role of Parvovirus B19 in non-immune hydrops fetalis at Hacettepe University, a major reference hospital in Turkey. Twenty-two cases of non-immune hydrops fetalis were retrospectively selected out of a total of 431 hydrops fetalis specimens from the Department of Pathology archieves. Paraffine embedded tissue sections from placental and liver tissues from each case were evaluated by histopathology, immunohistochemistry, nested PCR and commercial quantitative Real-time PCR. Viral DNA was detected in placental tissues by Real-time PCR in 2 cases (2/22, 9.1%) where histopathology also revealed changes suggestive of Parvovirus B19 infection. No significant histopathologic changes were observed for the remaining sections. Nested PCR that targets the VP1 region of the viral genome and immunohistochemistry for viral capsid antigens were negative for all cases. As a result, Parvovirus B19 is identified as the etiologic agent for the development of non-immune hydrops fetalis for 9.1% of the cases in Hacettepe University, Turkey. Real-time PCR is observed to be an effective diagnostic tool for nucleic acid detection from paraffine embedded tissues. Part of this study was presented as a poster at XIIIth International Congress of Virology, San Francisco, USA (Abstract V-572).

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Chondroitin 6-sulphate is required for neuroplasticity and memory in ageing

Molecular Psychiatry ◽

10.1038/s41380-021-01208-9 ◽

2021 ◽

Author(s):

Sujeong Yang ◽

Sylvain Gigout ◽

Angelo Molinaro ◽

Yuko Naito-Matsui ◽

Sam Hilton ◽

...

Keyword(s):

Chondroitin Sulphate ◽

Memory Loss ◽

Memory Deficits ◽

Long Term Potentiation ◽

Aged Mice ◽

Age Related ◽

Term Potentiation ◽

Early Memory

AbstractPerineuronal nets (PNNs) are chondroitin sulphate proteoglycan-containing structures on the neuronal surface that have been implicated in the control of neuroplasticity and memory. Age-related reduction of chondroitin 6-sulphates (C6S) leads to PNNs becoming more inhibitory. Here, we investigated whether manipulation of the chondroitin sulphate (CS) composition of the PNNs could restore neuroplasticity and alleviate memory deficits in aged mice. We first confirmed that aged mice (20-months) showed memory and plasticity deficits. They were able to retain or regain their cognitive ability when CSs were digested or PNNs were attenuated. We then explored the role of C6S in memory and neuroplasticity. Transgenic deletion of chondroitin 6-sulfotransferase (chst3) led to a reduction of permissive C6S, simulating aged brains. These animals showed very early memory loss at 11 weeks old. Importantly, restoring C6S levels in aged animals rescued the memory deficits and restored cortical long-term potentiation, suggesting a strategy to improve age-related memory impairment.

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Critical role of the CD44lowCD62Llow CD8+ T cell subset in restoring antitumor immunity in aged mice

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2103730118 ◽

2021 ◽

Vol 118 (23) ◽

pp. e2103730118

Author(s):

Yuka Nakajima ◽

Kenji Chamoto ◽

Takuma Oura ◽

Tasuku Honjo

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Critical Role ◽

Cell Subset ◽

Effector Memory ◽

T Cell Subset ◽

Aged Mice ◽

Age Related

CD8+ T cells play a central role in antitumor immune responses that kill cancer cells directly. In aged individuals, CD8+ T cell immunity is strongly suppressed, which is associated with cancer and other age-related diseases. The mechanism underlying this age-related decrease in immune function remains largely unknown. This study investigated the role of T cell function in age-related unresponsiveness to PD-1 blockade cancer therapy. We found inefficient generation of CD44lowCD62Llow CD8+ T cell subset (P4) in draining lymph nodes of tumor-bearing aged mice. In vitro stimulation of naive CD8+ T cells first generated P4 cells, followed by effector/memory T cells. The P4 cells contained a unique set of genes related to enzymes involved in one-carbon (1C) metabolism, which is critical to antigen-specific T cell activation and mitochondrial function. Consistent with this finding, 1C-metabolism–related gene expression and mitochondrial respiration were down-regulated in aged CD8+ T cells compared with young CD8+ T cells. In aged OVA-specific T cell receptor (TCR) transgenic mice, ZAP-70 was not activated, even after inoculation with OVA-expressing tumor cells. The attenuation of TCR signaling appeared to be due to elevated expression of CD45RB phosphatase in aged CD8+ T cells. Surprisingly, strong stimulation by nonself cell injection into aged PD-1–deficient mice restored normal levels of CD45RB and ameliorated the emergence of P4 cells and 1C metabolic enzyme expression in CD8+ T cells, and antitumor activity. These findings indicate that impaired induction of the P4 subset may be responsible for the age-related resistance to PD-1 blockade, which can be rescued by strong TCR stimulation.

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Ginsenoside Rb1 Ameliorates Age-Related Myocardial Dysfunction by Regulating the NF-κB Signaling Pathway

The American Journal of Chinese Medicine ◽

10.1142/s0192415x20500676 ◽

2020 ◽

Vol 48 (06) ◽

pp. 1369-1383

Author(s):

Shi-Ye Ke ◽

Ding-Hui Liu ◽

Lin Wu ◽

Xian-Guan Yu ◽

Min Wang ◽

...

Keyword(s):

Signaling Pathway ◽

Transforming Growth Factor ◽

Myocardial Dysfunction ◽

Internal Diameter ◽

Ginsenoside Rb1 ◽

Aged Mice ◽

Age Related ◽

Collagen Iii ◽

Related Proteins ◽

Molecular Expression

Age-related myocardial dysfunction is a very large healthcare burden. Here, we aimed to investigate whether ginsenoside Rb1 (Rb1) improves age-related myocardial dysfunction and to identify the relevant molecular mechanism. Young mice and aged mice were injected with Rb1 or vehicle for 3 months. Then, their cardiac function was inspected by transthoracic echocardiography. Serum and myocardium tissue were collected from all mice for histological or molecular expression analyses, including aging-related proteins, markers relevant to fibrosis and inflammation, and markers indicating the activation of the nuclear factor-kappa B (NF-[Formula: see text]B) pathway. Compared with the control condition, Rb1 treatment significantly increased the ejection fraction percentage and significantly decreased the internal diameter and volume of the left ventricle at the end-systolic and end-diastolic phases in aged mice. Rb1 treatment reduced collagen deposition and collagen I, collagen III, and transforming growth factor-[Formula: see text]1 protein expression levels in aged hearts. Rb1 also decreased the aging-induced myocardial inflammatory response, as measured by serum or myocardial interleukin-6 and tumor necrosis factor-[Formula: see text] levels. Furthermore, Rb1 treatment in aged mice increased cytoplasmic NF-[Formula: see text]B but decreased nuclear NF-[Formula: see text]B, which indicated the suppression of the NF-[Formula: see text]B signaling pathway by regulating the translocation of NF-[Formula: see text]B. Rb1 could alleviate aging-related myocardial dysfunction by suppressing fibrosis and inflammation, which is potentially associated with regulation of the NF-[Formula: see text]B signaling pathway.

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P5374Genetic deletion of IL-22 increased cardiac rupture after myocardial infarction in mice

European Heart Journal ◽

10.1093/eurheartj/ehz746.0337 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

M Yamamoto ◽

H Yasukawa ◽

J Takahashi ◽

S Nohara ◽

T Sasak ◽

...

Keyword(s):

Myocardial Infarction ◽

Real Time ◽

Real Time Pcr ◽

Tissue Repair ◽

Repair Process ◽

Cardiac Rupture ◽

Blue Dye ◽

Border Area ◽

Infarct Area

Abstract Background Interleukin-22 (IL-22) is a member of the IL-10 cytokine family, which mainly targets epithelial cells and does not target immune cells. Recently, it has been reported that IL-22 play roles in tissue repair in the skin and the liver; however, role of IL-22 in the process of tissue repair after myocardial infarction (MI) is unknown. Here, we investigated the role of IL-22 in tissue repair process after MI. Methods and results First, we examined the expression of IL-22 and its receptor IL-22RA1 in the wild type (WT) mice by real-time PCR. The expression of IL-22 and IL-22RA1 in the hearts were significantly increased 3 days after MI (p<0.05). To clarify the role of IL-22 in the heart after MI, we produced MI model in the WT mice and IL-22 knockout (KO) mice. We found that the IL-22 KO mice had significantly higher mortality than the WT mice after MI (p<0.05). Approximately 80% of the IL-22 KO mice died with cardiac rupture after MI. The infarct size which was estimated by evans blue dye and triphenyltetrazolium chloride staining at 3 days after MI was comparable between the IL-22 KO mice and the WT mice. Next, we performed real time PCR and PCR array analysis for tissue fibrosis and repair genes. We found that alpha-smooth muscle actin (aSMA), NF-kB, TNF-a and MMP13 (also known as collagenase-3) were significantly increased in the infarct area of IL-22 KO mice compared to WT mice. Immunostaining showed that the myofibroblast marker aSMA positive cells in the border area after MI were markedly higher in the IL-22 KO mice compared with the WT mice (p<0.05). Approximately 70% of cardiac rupture after MI in the IL-22 KO mice were occurred in the infarct area adjacent to the border area. Furthermore, we found aSMA positive cells and MMP13 positive cells around the ruptured site of the heart. Conclusion Thus, IL-22 KO mice exhibit high mortality and increased cardiac rupture after MI. And expression of aSMA and MMP13 were highly expressed in the ruptured site after MI in the IL-22 KO mice. These results suggest that IL-22 may play an important role in the tissue repair process after MI.

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128 THE ROLE OF ENDOTHELINS IN REGULATING BOVINE GRANULOSA CELLS STEROIDOGENESIS

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab128 ◽

2016 ◽

Vol 28 (2) ◽

pp. 194 ◽

Cited By ~ 1

Author(s):

L. F. Schütz ◽

J. E. Ervin ◽

L. Zhang ◽

C. Robinson ◽

M. Totty ◽

...

Keyword(s):

Growth Factor ◽

Real Time ◽

Granulosa Cells ◽

Real Time Pcr ◽

Linear Models ◽

Rna Extraction ◽

Insulin Like Growth Factor ◽

Steroid Production ◽

Oestradiol Production

Endothelins are a group of vasoactive 21 amino acid peptides reported to play roles in steroidogenesis, folliculogenesis, and ovulation (Bridges et al. 2012 Life Sci. 91, 501–506). Nevertheless, the role of endothelins in regulating steroidogenesis in the bovine species requires further investigation. Thus, the objective of this study was to investigate the effects of endothelin 1 (ET-1) and endothelin 2 (ET-2) on bovine granulosa cell (GC) steroidogenesis. Bovine ovaries were obtained from a local abattoir. Follicular fluid was aspirated from small (1–5 mm) follicles and GC were isolated and exposed to various treatments (ET-1, ET-2, or ET-1 plus ET-2 with FSH and with or without insulin-like growth factor-1). In replicated experiments, culture medium was removed and analysed for steroid production via radioimmunoassay. Granulosa cells were either harvested with trypsin and counted using a Coulter Counter or collected with Trizol for RNA extraction and quantification via real-time PCR (18S rRNA was used as a housekeeping gene). Steroid production was expressed as nanograms (in the case of progesterone) and picograms (in the case of oestradiol) per 105 cells per 24 h. Relative quantity of target gene mRNA was expressed as 2–ΔΔCt using the relative comparative threshold cycle (Ct) method. Data were analysed via ANOVA and the general linear models (GLM) procedure of SAS for Windows (SAS Institute Inc., Cary, NC). If a significant main effect was identified, differences among means were determined by Fisher’s protected least significant differences test. The values were reported as least squares means ± standard error of the mean. In the presence of insulin-like growth factor-1, ET-1 significantly inhibited oestradiol production at 300 ng mL–1 (100.30 ± 11.05; P < 0.05), but not at 30 ng mL–1 (114.47 ± 11.05; P > 0.05) in comparison to the control (141.21 ± 11.05), whereas no differences were observed for progesterone production at 300 ng mL–1 (60.11 ± 7.11; P > 0.05) or at 30 ng mL–1 (64.02 ± 7.11; P > 0.05) in comparison to control (76.75 ± 7.11). ET-2 also significantly inhibited oestradiol production at 300 ng mL–1 (91.08 ± 11.87; P < 0.01), but not at 30 ng mL–1 (112.77 ± 11.87; P > 0.05) in comparison to the control in the presence of insulin-like growth factor-1. No significant effect of ET-1 and ET-2 was observed on steroidogenesis of granulosa cells cultured without insulin-like growth factor-1. Consistent with steroids production data, real-time PCR results indicated that, in the presence of IGF-1, ET-1 (5.66 ± 1.05) and ET-2 (5.65 ± 1.05) inhibited (P < 0.05) aromatase gene expression compared to controls (11.33 + 1.05), and ET-1 plus ET-2 (2.42 ± 1.05) reduced (P < 0.05) expression below that observed with either alone. No effect of ET-1 (4.38 ± 0.95; P > 0.05), ET-2 (5.94 ± 0.95; P > 0.05), or ET-1 plus ET-2 (4.57 ± 0.95; P > 0.05) was observed for side-chain cleavage enzyme (CYP11A1) in comparison to controls (4.4 ± 1.07). Altogether, these results indicate that endothelins are involved in the regulation of steroidogenesis of bovine GC.

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