Thy-1 Expression Research Articles

Thy1 (CD90) Expression Is Elevated in Radiation-Induced Periprosthetic Capsular Contracture: Implication for Novel Therapeutics.

Capsular contracture is a devastating complication of postmastectomy implant-based breast reconstruction. Unfortunately, capsular contracture rates are drastically increased by targeted radiotherapy, a standard postmastectomy treatment. Thy1 (also called CD90) is important in myofibroblast differentiation and scar tissue formation. However, the impact of radiotherapy on Thy1 expression and the role of Thy1 in capsular contracture are unknown. The authors analyzed Thy1 expression in primary human capsular tissue and primary fibroblast explants by real-time quantitative polymerase chain reaction, Western blotting, and immunohistochemistry. Thy1 was depleted using RNA interference to determine whether Thy1 expression was essential for the myofibroblast phenotype in capsular fibroblasts. Furthermore, human capsular fibroblasts were treated with a new antiscarring compound, salinomycin, to determine whether Thy1 expression and myofibroblast formation were blocked by salinomycin. In this article, the authors show that radiation therapy significantly increased Thy1 mRNA and protein expression in periimplant scar tissue. Capsular fibroblasts explanted from scar tissue retained the ability to make the myofibroblast-produced scar-forming components collagen I and α-smooth muscle actin. Depletion of Thy1 decreased the fibrotic morphology of capsular fibroblasts and significantly decreased α-smooth muscle actin and collagen levels. Furthermore, the authors show for the first time that salinomycin decreased Thy1 expression and prevented myofibroblast formation in capsular fibroblasts. These data reveal that ionizing radiation-induced Thy1 overexpression may contribute to increased capsular contracture severity, and fibroblast scar production can be ameliorated through targeting Thy1 expression. Importantly, the authors' new results show promise for the antiscarring ability of salinomycin in radiation-induced capsular contracture. Therapeutic, V.

Therapeutic efficacy of neural stem cells originating from umbilical cord-derived mesenchymal stem cells in diabetic retinopathy

The aim of this study was to evaluate the effects of intravitreal injection of neural stem cells (NSCs) originating from human umbilical cord-derived mesenchymal stem cells (UC-MSCs) on neurodegeneration of diabetic retinopathy (DR) in rats. UC-MSCs were isolated and passaged, followed by induction to NSCs in neural differentiation medium. Four weeks following NSC transplantation, treatment attenuated retinal vascular dysfunction compared with non-treated rats, and BDNF and Thy-1 expression was significantly higher in the treated group than in the control group. Treatment of diabetic rats with NSCs prevented the decrease in BDNF levels caused by diabetes. The average leakage of Evans Blue (EB) dye in the treated group was significantly less than that in the control group. These morphological improvements were accompanied by a restoration of vision, as documented by F-ERG. NSCs originating from MSCs demonstrated a neuroprotective effect by increasing the number of surviving RGCs and significantly reducing the progression of DR. Thus, transplantation of NSCs could be a novel strategy for the treatment of neurodegeneration in DR.

Portal fibroblasts marked by the surface antigen Thy1 contribute to fibrosis in mouse models of cholestatic liver injury.

Liver fibrosis, a condition that is characterized by excessive production and accumulation of extracellular matrix, including collagen, is the most common outcome of chronic liver injuries of different etiologies. Vitamin A‐storing hepatic stellate cells (HSCs) are considered to be the main source of this collagen production, with activation in response to liver injury. In contrast, the contribution of other cell types to this fibrogenic response remains largely elusive due to the lack of specific surface markers to identify and isolate these cells for detailed analysis. Here, we identify a mesenchymal population of thymus cell antigen 1 (Thy1)+ CD45− cells (Thy1 MCs) in the mouse liver; these cells reside near the portal vein in vivo and indicate profibrogenic characteristics in vitro, shown by their expression of collagen and α‐smooth muscle actin. Flow cytometric analysis of mouse liver nonparenchymal cells revealed that vitamin A storage and Thy1 expression were mutually exclusive, indicating that Thy1 MCs are distinct from HSCs. Importantly, Thy1 MCs reacted and contributed to the development of liver fibrosis specifically in mouse models of cholestatic liver injury. With the occurrence of cholestatic liver injury, collagen‐producing Thy1 MCs expanded in cell number and inhibited collagen degradation through up‐regulation of matrix metalloproteinase inhibitor Timp1 expression, thereby promoting the accumulation of extracellular matrix in the periportal area. Conclusion: This study establishes Thy1 as a useful cell surface marker to prospectively identify and isolate periportal fibroblasts and further highlights a significant contribution of these cells to the pathogenesis of liver fibrosis caused by cholestatic liver injuries. We suggest that Thy1 MCs may be an interesting therapeutic target for treating liver fibrosis in addition to the well‐characterized HSCs. (Hepatology Communications 2017;1:198‐214)

Thy-1 interaction with Fas in lipid rafts regulates fibroblast apoptosis and lung injury resolution

Thy-1-negative lung fibroblasts are resistant to apoptosis. The mechanisms governing this process and its relevance to fibrotic remodeling remain poorly understood. By using either sorted or transfected lung fibroblasts, we found that Thy-1 expression is associated with downregulation of anti-apoptotic molecules Bcl-2 and Bcl-xL, as well as increased levels of cleaved caspase-9. Addition of rhFasL and staurosporine, well-known apoptosis inducers, caused significantly increased cleaved caspase-3, -8, and PARP in Thy-1-transfected cells. Furthermore, rhFasL induced Fas translocation into lipid rafts and its colocalization with Thy-1. These in vitro results indicate that Thy-1, in a manner dependent upon its glycophosphatidylinositol anchor and lipid raft localization, regulates apoptosis in lung fibroblasts via Fas-, Bcl-, and caspase-dependent pathways. In vivo, Thy-1 deficient (Thy1−/−) mice displayed persistence of myofibroblasts in the resolution phase of bleomycin-induced fibrosis, associated with accumulation of collagen and failure of lung fibrosis resolution. Apoptosis of myofibroblasts is decreased in Thy1−/− mice in the resolution phase. Collectively, these findings provide new evidence regarding the role and mechanisms of Thy-1 in initiating myofibroblast apoptosis that heralds the termination of the reparative response to bleomycin-induced lung injury. Understanding the mechanisms regulating fibroblast survival/apoptosis should lead to novel therapeutic interventions for lung fibrosis.

Editor's Highlight: Thy1 (CD90) Expression is Reduced by the Environmental Chemical Tetrabromobisphenol-A to Promote Adipogenesis Through Induction of microRNA-103.

Environmental chemicals termed "obesogens" disrupt the endocrine system to promote adipogenesis and obesity. Tetrabromobisphenol-A (TBBPA) has been reported to increase adipogenesis; however, the mechanism(s) of action are unclear. Thy1 (CD90) is a glycophosphatidylinositol-anchored membrane protein that serves as a marker for stem cells and also plays an important role in regulating adipogenesis and obesity. We investigated whether or not TBBPA promotes adipogenesis in human and mouse cells by reducing Thy1 levels. We further sought to identify the molecular mechanism(s) whereby TBBPA targets Thy1 expression. Mouse and human cells were exposed to TBBPA, and Thy1 expression was analyzed using flow cytometry, Western blotting, and qPCR. We tested whether microRNAs predicted to target Thy1 (miR-103 and miR-107) were upregulated by TBBPA using quantitative PCR assays. We also determined if Thy1 mRNA was a bona fide miR-103/107 target. Our results show that Thy1 expression was reduced in both human and mouse cells after exposure to TBBPA. Both Thy1 mRNA and protein levels were decreased by low-dose TBBPA exposure. TBBPA reduced Thy1 levels and further increased adipogenesis when an adipogenic medium was used. Mechanistically, we show that miR-103 and miR-107 are induced by TBBPA and that miR-103 targets Thy1 to reduce its expression. Our results reveal for the first time that Thy1 is a target of TBBPA. Furthermore, our data support the concept that Thy1 is a key marker targeted by environmental chemicals that promote adipogenesis and obesity.

Analysis of melanopsin gene expression in the rabbit retina at different ages.

Melanopsin is the photopigment of intrinsically photosensitive retinal ganglion cells that mediate non-visual responses to light. The aim of this study was to describe and analyze melanopsin gene expression in the rabbit retina at different ages and compare its expression with the prototypic gene of retinal ganglion cells (Thy-1 gene). Expression levels of OPN4, Thy-1, and GADPH genes were measured by real-time PCR at 3, 4, 8, 11, 12, 17, 19, 20, 23, 27, 32, and 47 postnatal days. We also regrouped the days before and after day 12 of life (pre-photic and post-photic stage, respectively). Average expression of the OPN4 gene between days was similar (P = 0.713), but was statistically different in the Thy-1 gene (P = 0.004). Also, no significant differences were found in OPN4 gene expression pre-photic and post-photic stage (P = 0.629); however, Thy-1 expression was higher in the pre-photic stage, almost double, than in the post-photic stage, with significant differences (P = 0.001). This is the first report describing OPN4 gene expression in the rabbit retina at different ages. We demonstrated that the OPN4 gene is constantly expressed at all early stages, even before the onset of photoentrainment by the pups and that Thy-1 and OPN4 gene expressions are out of phase.

Negligible Oval Cell Proliferation Following Ischemia-Reperfusion Injury With and Without Partial Hepatectomy.

Hepatic oval cells proliferate to replace hepatocytes and restore liver function when hepatocyte proliferation is compromised or inadequate. Exposure to chemical carcinogens, severe liver steatosis, and partial hepatectomy has been used in animal models to demonstrate the role of oval cells in liver regeneration. Ischemia-reperfusion injury (IRI) causes hepatocellular damage and death in the absence of confounding chemical toxicity; however, oval cell induction by IRI has not been demonstrated in vivo. We examine oval cell induction following partial IRI. Wistar rats were subjected to 2 IRI protocols: 70% warm liver ischemia for 30 minutes followed by reperfusion or 70% warm liver ischemia for 30 minutes with partial hepatectomy of the nonischemic lobes followed by reperfusion. Liver injury was monitored by serum alanine aminotransferase (ALT) at 1 day and 7 days of reperfusion. Oval cell proliferation was monitored by indirect immunofluorescence staining using the surface markers BD.2 and Thy-1. Cellular proliferation was quantified by 5-ethynyl-2'-deoxyuridine (EdU) incorporation in vivo. Serum ALT elevation was only observed at the 1-day time point in the IRI with partial hepatectomy model. Oval cell marker expression was restricted to the biliary structures in both the ischemic and the nonischemic control lobes. Oval cell induction, measured by changes in the frequency of BD.2 and Thy-1 expression and EdU incorporation, was not significantly altered by IRI. In both mild and moderate IRI models, we did not find evidence of oval cell induction or proliferation. EdU staining was restricted to hepatocytes, suggesting that liver regeneration following IRI is mediated by hepatocyte proliferation.

Brain-derived neurotrophic factor and retinal ganglion cells in the retina of diabetic rat after intravitreal injection of human umbilical cord mesenchymal stem cells-induced neural stem cells

Objective To investigate the effect of intravitreal injection of neural stem cells (NSC) derived from human umbilical cord mesenchymal stem cells (hUCMSC) on the expression of brain-derived neurotrophic factor (BDNF) and the number of retinal ganglion cells (RGC). Methods Fifty-two adult male Sprague-Dawley rats were randomly divided into normal group (group A) and diabetes mellitus group which received intraperitoneal injection of streptozocin to make diabetic rat models. One month after the diabetic rat models were confirmed successfully, diabetic rats were randomly divided into diabetic group (group B), hUCMSC group (group C) and hUCMSC-induced NSC group (group D). And thirteen diabetic rats were included in each group. Immuno-cytochemistry was applied to observe BDNF and thymosin-1(Thy-1) staining in the retina. Then mean integrated absorbance of the staining region on the retina slices were analyzed by Image-Pro Plus 6.0. The number of Thy-1 labeled RGC was record. Results BDNF and Thy-1 were positive on the retina slices from group A. The staining intensity from group B became weak and the expression of BDNF and Thy-1 gradually decrease with time (P 0.05) at 2 weeks after injection, but were significant different for other time points (P<0.05). Significant positive correlation between the expression of BDNF and the number of RGC were found by the Pearson correlation analysis (r=0.964, P<0.05). Conclusion Intravitreal injection of hUCMSC-derived NSC to diabetic rat may protect the retina by promoting the expression of BDNF and increasing the number of RGC. Key words: Diabetic retinopathy/therapy; Neural stem cells/transplantation; Brain-derived neurotrophic factor; Retinal ganglion cells; Animal experimentation

Overexpression of Thy1 and ITGA6 is associated with invasion, metastasis and poor prognosis in human gallbladder carcinoma.

Gallbladder cancer (GBC) is a rare but highly aggressive cancer for which no well-accepted prognostic biomarkers have been identified. Thymus cell antigen 1 (Thy1), also known as cluster of differentiation (CD)90, and integrin α6 (ITGA6), also known as CD49f, are important molecules in cancer and putative markers of various stem cell types. However, their role in GBC remains to be elucidated. In the present study, Thy1 and ITGA6 expression status in clinical GBC samples, which comprised squamous cell/adenosquamous carcinoma (SC/ASC) and adenocarcinoma (AC) subtypes, was investigated. The associations between Thy1 and ITGA6 expression and clinical parameters and survival rate were analyzed separately. The THY1 and ITGA6 messenger RNA levels were significantly higher in both SC/ASC and AC tissues than in adjacent non-tumor tissues (all P<0.001). These results were subsequently confirmed by immunohistochemical analyses. Overexpression of Thy1 and ITGA6 was correlated with poor differentiation, large tumor size, lymph node metastasis and great invasiveness in SC/ASC (Thy1, P=0.045, P=0.005, P=0.003 and P=0.009, respectively, and ITGA6, P=0.029, P=0.011, P=0.009 and P=0.004, respectively) and AC (Thy1, P=0.027, P<0.001, P=0.003 and P 0.004, respectively, and ITGA6, P=0.002, P=0.003, P=0.006 and P=0.006, respectively). Both Thy1 and ITGA6 were expressed at higher levels in AC with advanced tumor-node-metastasis stage (TNM) than in AC with low TNM stage (P=0.001 and P=0.018, respectively). In addition, patients with elevated Thy1 or ITGA6 expression had shorter overall survival than those with negative Thy1 or ITGA6 expression. Multivariate Cox regression analysis demonstrated that Thy1 (SC/ASC, P=0.001 and AC, P=0.005) and ITGA6 (both P=0.003) were independent predictors of poor prognosis in both SC/ASC and AC patients. In conclusion, Thy1 and ITGA6 could be clinical prognostic markers for GBC.

Lipopolysaccharide promotes pulmonary fibrosis in acute respiratory distress syndrome (ARDS) via lincRNA-p21 induced inhibition of Thy-1 expression.

Acute respiratory distress syndrome (ARDS) is a common clinical disorder characterized by pulmonary edema leading to acute lung damage and arterial hypoxemia. Pulmonary fibrosis is a progressive, fibrotic lung disorder, whose pathogenesis in ARDS remains speculative. LincRNA-p21 was a novel regulator of cell proliferation, apoptosis and DNA damage response. This study aims to investigate the effects and mechanism of lincRNA-p21 on pulmonary fibrosis in ARDS. Purified 10mg/kg LPS was dropped into airways of C57BL/6 mice. Expression levels of lincRNA-p21 and Thy-1 were measured by real-time PCR or western blotting. Proliferation of lung fibroblasts was analyzed by BrdU incorporation assay. Lung and BAL collagen contents were estimated using colorimetric Sircol assay. LincRNA-p21 expression was time-dependently increased and Thy-1 expression was time-dependently reduced in a mouse model of ARDS and in LPS-treated lung fibroblasts. Meanwhile, lung fibroblast proliferation was also time-dependently elevated in LPS-treated lung fibroblasts. In addition, lung fibroblast proliferation could be promoted by lincRNA-p21 overexpression and LPS treatment, however, the elevated lung fibroblast proliferation was further abrogated by Thy-1 overexpression or lincRNA-p21 interference. And Thy-1 interference could elevate cell viability of lung fibroblasts and rescue the reduction of lung fibroblast proliferation induced by lincRNA-p21 interference. Moreover, lincRNA-p21 overexpression dramatically inhibited acetylation of H3 and H4 at the Thy-1 promoter and Thy-1 expression levels in HLF1 cells. Finally, lincRNA-p21 interference rescued LPS-induced increase of lung and BAL collagen contents. LincRNA-p21 could lead to pulmonary fibrosis in ARDS by inhibition of the expression of Thy-1.

The clinical pathological significance of Thy1 and CD49f expression in chondrosarcomas

ObjectiveThis study investigated the protein expression and clinicopathological significance of Thy1 and CD49f in chondrosarcomas. MethodsThy1 and CD49f protein expression in 59 chondrosarcomas and 33 osteochondromas were measured by immunohistochemical staining. ResultsThe percentage of positive Thy1 and CD49f expression was significantly higher in patients with chondrosarcoma than in patients with osteochondroma (P<0.01). The percentage of positive Thy1 and CD49f expression was significantly lower in patients with histological grade I, Enneking stage I, AJCC stage I/II stage, non-metastatic and non-invasive chondrosarcoma than in patients with histological grade III, Enneking stage II+III, AJCC stage III/IV, metastatic and invasive chondrosarcoma (P<0.05 or P<0.01). Thy1 expression was positively correlated with CD49f expression in chondrosarcoma. Kaplan-Meier survival analysis showed that histological grade, AJCC stage, Enneking stage, metastasis, invasion, and Thy1 and CD49f expression significantly correlated with shorter mean survival time in chondrosarcoma patients (P<0.05 or P<0.01). Cox multivariate analysis showed that positive Thy1 and CD49f expression was an independent prognostic factor that negatively correlated with overall postoperative survival. ConclusionPositive Thy1 and CD49f expression is significantly associated with the progression and poor prognosis of chondrosarcoma.

Expansion and Hepatic Differentiation of Adult Blood-Derived CD34+ Progenitor Cells and Promotion of Liver Regeneration After Acute Injury

The low availability of functional hepatocytes has been an unmet demand for basic scientific research, new drug development, and cell-based clinical applications for decades. Because of the inability to expand hepatocytes in vitro, alternative sources of hepatocytes are a focus of liver regenerative medicine. We report a new group of blood-derived CD34(+) progenitor cells (BDPCs) that have the ability to expand and differentiate into functional hepatocyte-like cells and promote liver regeneration. BDPCs were obtained from the peripheral blood of an adult mouse with expression of surface markers CD34, CD45, Sca-1, c-kit, and Thy1.1. BDPCs can proliferate in vitro and differentiate into hepatocyte-like cells expressing hepatocyte markers, including CK8, CK18, CK19, α-fetoprotein, integrin-β1, and A6. The differentiated BDPCs (dBDPCs) also display liver-specific functional activities, such as glycogen storage, urea production, and albumin secretion. dBDPCs have cytochrome P450 activity and express specific hepatic transcription factors, such as hepatic nuclear factor 1α. To demonstrate liver regenerative activity, dBDPCs were injected into mice with severe acute liver damage caused by a high-dose injection of carbon tetrachloride (CCl4). dBDPC treatment rescued the mice from severe acute liver injury, increased survival, and induced liver regeneration. Because of their ease of access and application through peripheral blood and their capability of rapid expansion and hepatic differentiation, BDPCs have great potential as a cell-based therapy for liver disease. Hematopoietic stem/progenitor cell expansion and tissue-specific differentiation in vitro are challenges in regenerative medicine, although stem cell therapy has raised hope for the treatment of liver diseases by overcoming the scarcity of hepatocytes. This study identified and characterized a group of blood-derived progenitor cells (BDPCs) from the peripheral blood of an adult mouse. The CD34(+) progenitor-dominant BDPCs were rapidly expanded and hepatically differentiated into functional hepatocyte-like cells with our established coculture system. BDPC treatment increased animal survival and produced full regeneration in a severe liver injury mouse model caused by CCl4. BDPCs could have potential for liver cell therapies.

Hypothermia Protects and Prolongs the Tolerance Time of Retinal Ganglion Cells against Ischemia.

PurposeHypothermia has been shown to be neuroprotective in the therapy of ischemic stroke in the brain. To date no studies exist on the level of the inner retina and it is unclear if hypothermia would prolong the ischemic tolerance time of retinal ganglion cells, which are decisive in many ischemic retinopathies.MethodsBovine eyes were enucleated and stored either at 21°C or 37°C for 100 or 340 minutes, respectively. Afterwards the globes were dissected, the retina was prepared and either the spontaneous ganglion cell responses were measured or the retina was incubated as an organotypic culture for additional 24 hours. After incubation the retina was either processed for histology (H&E and DAPI staining) or real-time PCR (Thy-1 expression) was performed.ResultsHypothermia prolonged ganglion cell survival up to 340 minutes under ischemic conditions. In contrast to eyes kept at 37°C the eyes stored at 21°C still showed spontaneous ganglion cell spiking (56.8% versus 0%), a 5.8 fold higher Thy-1 mRNA expression (not significant, but a trend) and a preserved retinal structure after 340 minutes of ischemia.ConclusionHypothermia protects retinal ganglion cells against ischemia and prolongs their ischemic tolerance time.

Effect and mechanism of inhibition of lipopolysaccharide-induced pulmonary fibrosis by butyric acid

To evaluate the inhibitory effect of butyric acid (BA) as a histone deacetylase (HDAC) inhibitor on lipopolysaccharide (LPS)-induced pulmonary fibrosis and its mechanism. Thirty C57/BL6 mice were randomly divided into three groups according to the random number method, namely control group (physiological saline was given intraperitoneally and by gavage), LPS challenge group (LPS-induced murine model of pulmonary fibrosis was reproduced with intraperitoneal injection of 10 mg/kg LPS), and BA preconditioning + LPS challenge group (10 mg/kg BA was given followed by intraperitoneal injection of 10 mg/kg LPS), with 10 mice in each group. Mice were sacrificed painlessly, and lung tissue samples were harvested at 2 weeks and 4 weeks respectively (five samples every group each time). HDAC activity was evaluated with fluorescence analysis kit. Protein expression of acetylated-histone H3 (Ace-H3), acetylated-histone H4 (Ace-H4) and thymocyte differentiation antigen 1 (Thy-1) were determined by Western Blot. The mRNA expression of Thy-1 was assessed by real-time reverse transcription- polymerase chain reaction (real-time RT-PCR). The degree of lung inflammation and fibrosis were microscopic detected after hematoxylin-eosin (HE) staining and Masson collagen staining. The deposition of lung collagen was detected by hydroxyproline content measurement kit. Compared to control group, the degree of lung inflammation and fibrosis was aggravated after LPS challenge, as manifested by increased hydroxyproline content (μg/mg, 2 weeks: 8.384±0.632 vs. 4.388±0.334, 4 weeks: 8.308±0.244 vs. 4.370±0.342, both P < 0.01), increased HDAC activity (μmol/L, 2 weeks: 7.243±0.384 vs. 3.628±0.641, 4 weeks: 6.479±0.202 vs. 3.238±0.524, both P < 0.01), increased deacetylation degree of histone H3 and H4 [relative expression of Ace-H3 (gray value): 0.516±0.115 vs. 1.005±0.359 at 2 weeks, 0.633±0.143 vs. 1.092±0.193 at 4 weeks, both P < 0.05; relative expression of Ace-H4 (gray value): 0.402±0.164 vs. 0.759±0.187 at 2 weeks, P > 0.05; 0.426±0.098 vs. 0.858±0.177 at 4 weeks, P < 0.01], and lowered Thy-1 mRNA and protein expression [Thy-1 mRNA (2(-ΔΔCt)): 0.606±0.066 vs. 1.005±0.109 at 2 weeks, P < 0.01; 0.824±0.101 vs. 1.210±0.400 at 4 weeks, P > 0.05; relative expression of Thy-1 protein (gray value): 0.725±0.284 vs. 1.249±0.297 at 2 weeks, 0.589±0.139 vs. 1.372±0.343 at 4 weeks, both P < 0.05]. Compared with LPS group, BA precondition could inhibit above processes, as manifested by decreased hydroxyproline content (μg/mg: 5.943±0.726 vs. 8.384±0.632 at 2 weeks, 4.938±0.209 vs. 8.308±0.244 at 4 weeks, both P < 0.01), decreased HDAC activity (μmol/L: 4.386±0.117 vs. 7.243±0.384 at 2 weeks, 4.863±0.096 vs. 6.479±0.202 at 4 weeks, both P < 0.01), increased Thy-1 mRNA expression at 2 weeks (2(-ΔΔCt): 0.884±0.216 vs. 0.606±0.066, P < 0.05), increased acetylation degree of histone H4 and Thy-1 protein expression at 4 weeks [relative expression of Ace-H4 (gray value): 0.715±0.145 vs. 0.426±0.098, P < 0.05; relative protein expression of Thy-1 (gray value): 0.939±0.098 vs. 0.589±0.139, P < 0.01]. LPS-induced pulmonary fibrosis was related with activation of HDAC, deacetylation of histone H3 and H4 and Thy-1 gene silencing. HDAC inhibitor BA could inhibit LPS-induced pulmonary fibrosis and Thy-1 gene silencing through inhibiting activation of HDAC and deacetylation of histone H4.

Restrain of hypoxia on the expression of annexin A2 in mouse RGC-5 cells

Background Hypoxia is the main factor of retinal neovascularization and is closely associated with retinal ganglial cells (RGCs) degeneration.However, the study of retinal neural tissue lesions is rare. Objective This study was to investigate the influence of hypoxia environment on the expression of annexin A2 (ANXA2) in mouse RGC-5 cells and explore the mechanism of RGCs damage induced by hypoxia. Methods Immortalized mouse RGC-5 cells were cultured in high glucose DMEM with 10% fetal bovine serum.The cells were identified by detecting the expression of Thy-1, a specific biomarker of RGCs.CoCl2 was added into the medium at the final concentrations of 50, 100, 200 and 300 μmol/L, and the cells without CoCl2 served as the control group.Cell viability (absorbance) was assayed by cell counting kit-8 (CCK-8) method in 12, 24 and 48 hours after addition of CoCl2.The hypoxic cell models were established in DMEM with 100 μmol/L CoCl2 and divided into the hypoxic 3-hour group, hypoxic 6-hour group, hypoxic 12-hour group and hypoxic 24-hour group, with the normal cultured cells as the normal control group.Apoptotic cells were determined by using hoechst 33342 stain.The expression levels of ANXA2 mRNA and protein in the cells were detected by real-time quantification PCR and Western blot, respectively.The expression and location of ANXA2 in the cells were examined by using immunofluorescence technique. Results The cultured cells grew well and showed the fusiform and polygonal shape, with positive expression of Thy-1 protein.Compared with the normal control group, the viabilities of the cells were insignificantly changed in the 50 μmol/L CoCl2 group and 100 μmol/L CoCl2 group (all at P>0.05), but the cell viabilities were significantly reduced in the 200 μmol/L CoCl2 group and 300 μmol/L CoCl2 group in various time points (all at P<0.05). Hoechst 33342 staining showed that the apoptotic cells with nuclear condensation and high green fluorescence intensity were obtained in the hypoxia groups.The relative expression levels of ANXA2 mRNA were significantly lower in the hypoxic groups than those in the normal control group (all at P<0.05). The relative expression levels of ANXA2 protein were significantly lower in the hypoxia 3-, 6-, 12- and 24-hour group than those in the normal control group (all at P<0.05). Apoptotic cells were seen in the hypoxic 3-hour group, hypoxic 6-hour group, hypoxic 12-hour group and hypoxic 24-hour group compared with the normal control group, showing the bright blue fluorescence in cellular nucleus for hoechst 33342.The relative expressing levels of ANXA2 mRNA in the cells were 0.80±0.14, 0.67±0.33, 0.49±0.17 and 0.39±0.02 in the hypoxic 3-hour group, hypoxic 6-hour group, hypoxic 12-hour group and hypoxic 24-hour group, which were significantly declined in comparison with the normal control group, with a statistically difference among the groups (F=434.354, P=0.000). The relative expression values of ANXA2 protein were 0.552 6±0.012 3, 0.425 9±0.033 4, 0.344 9±0.017 8 and 0.382 7±0.022 1 in the hypoxic 3-hour group, hypoxic 6-hour group, hypoxic 12-hour group and hypoxic 24-hour group, which were remarkably lower than 0.602 1±0.001 4 in the normal control group, showing considerably difference among the groups (F=3.057, P=0.000). ANXA2 proteins were highly expressed in the cellular nucleus and less expressed in the cell membrane and cytoplasm in the normal cells.Compared with the normal control group, the ANXA2 protein showed weak expression in the hypoxia group and primarily in the cytoplasm. Conclusions The expression of ANXA2 down-regulates in hypoxic mouse RGC-5 cells, which may participate in the apoptosis process of RGCs in high glucose environment. Key words: Annexin A2; Retinal ganglion cells; Hypoxia; Apoptosis

230 EFFECT OF DIFFERENT CONCENTRATIONS OF GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR ON EXPRESSION OF SELF-RENEWAL RELATED GENES IN GOAT (CAPRA HIRCUS) SPERMATOGONIAL STEM CELLS

Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor-β superfamily produced by Sertoli cells, is essential for the self-renewal of spermatogonial stem cells in vivo. The present study evaluated the effects of different concentrations of GDNF (human recombinant expressed in Escherichia coli) on expression of some self-renewal related genes in spermatogonial stem cells (SSC). The SSC were isolated from prepubertal goat testes (3–6 months of age) by using double enzymatic digestion method and filtration through 80- and 60-µm nylon mesh filters. Further enrichment was achieved by differential plating on Datura stramonium agglutinin (DSA) lectin-coated dishes and Percoll density gradient centrifugation. The enriched cells were cultured on goat Sertoli cell feeder layer in DMEM + 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Primary SSC colonies were formed within 7 to 10 days. These colonies were characterised through alkaline phosphatase and immunofluorescence staining on Day 10 for different SSC-specific protein markers. Colonies were found to be positive for DBA, THY1, PLZF, UCHL1, OCT-4, SOX2, and NANOG, and negative for c-Kit expression. These colonies were cultured for 15 days without or with supplementation of GDNF forming following groups: (1) without GDNF (control), (2) 10 ng mL–1 GDNF, (3) 20 ng mL–1 GDNF, and (4) 40 ng mL–1 GDNF. RNA was isolated from 100 colonies from 3 different trials on Day 15 of culture, and relative expression of different self-renewal related genes was determined by qRT-PCR. Relative mRNA abundance of PLZF was higher (P &lt; 0.05) following supplementation with 40 ng mL–1 GDNF than in other groups (i.e. control, 10 and 20 ng mL–1 GDNF). Expression of BCL6B and ID4 was found to be significantly higher (P &lt; 0.05) after supplementation of GDNF at all concentrations compared with the control group. Expression of UCHL1 was higher with addition of 20 and 40 ng mL–1 GDNF (P &lt; 0.05), whereas expression of THY1 was higher with supplementation of 10 ng mL–1 GDNF (P &lt; 0.05). In conclusion, GDNF was found to benefit expression of goat SSC candidate genes at a concentration of 40 ng mL–1.

221 DERIVATION OF INSULIN-PRODUCING CELLS FROM HUMAN DERMAL FIBROBLASTS AT INTERMEDIATE PHASE OF REPROGRAMMING

Process of somatic cell reprogramming into induced pluripotent stem (iPS) cells can broadly be divided into an early stochastic phase and a later, hierarchical and more deterministic phase of gene activation. Between the stochastic onset and the deterministic progression while approaching pluripotency, cells upon reprogramming undergo a transient activation or repression of developmental regulators. In these intermediate cells yet acquiring pluripotency, many somatic gene expressions are lost, while various lineage-specific factors are activated. In the present study, we produced pancreatic β cells by exposing human dermal fibroblasts (HDF) at the intermediate phase of reprogramming to pancreas-specific culture conditions. To obtain the intermediate cells, reprogramming factor Oct4, Sox2, Klf4, and c-Myc were transduced into HDF using retroviruses. By Day 9 after transduction, the expression of fibroblast-associated Thy1 was lost, while the early reprogramming marker CD49d was activated, suggesting that the cells are at the early stage of intermediate phase. These cells were capable of differentiating into 3 germ layers when cultured in tri-differentiation medium (including 100 ng mL–1 Activin A, 0.1 ng mL–1 Wnt3 for endodermal differentiation; 20 ng mL–1 BMP4, 20 ng mL–1 Activin A for mesodermal differentiation; 10 μM SB431542, 600 ng mL–1 Dorsomorphin, and 35 ng mL–1 Noggin for ectodermal differentiation) and expressing lineage-specific markers Sox17 (endoderm), Brachyury (mesoderm), and Otx2 (ectoderm). Further, the endodermal cells derived from HDF at intermediate phase of reprogramming were cultured in pancreas-induction medium (KO DMEM with 1% insulin-transferrin-selenium (ITS), 10 ng mL–1 BMP4, 10 mM nicotinamide, 10 μM forskolin, 10 μM dexamethasone, and 50 ng mL–1 exendin-4). After 5 days in culture, cells expressed pancreatic progenitor marker Pdx1, as examined by immunostaining and fluorescence-activated cell sorting (FACS) analysis. After culture for additional 10 days, the cells formed spherical clusters and expressed pancreatic islet-specific genes including insulin, somatostatin, glucagon, Nkx6.1, and MafA, as determined by RT-PCR. Moreover, the production of insulin and C-peptide from the derived β cells was confirmed by immunocytochemistry and FACS analysis. In response to glucose stimulation, these cells secreted insulin and C-peptide, as measured by ELISA. The present study demonstrates that fully differentiated functional cells could be converted from somatic cells at intermediate phase of reprogramming by exposing cells to specific culture conditions. The results shown in this study may provide a faster and potentially safer way to produce functional pancreatic cells without isolation and differentiation of iPS cells.

Conformational coupling of integrin and Thy-1 regulates Fyn priming and fibroblast mechanotransduction

Progressive fibrosis is characterized by excessive deposition of extracellular matrix (ECM), resulting in gross alterations in tissue mechanics. Changes in tissue mechanics can further augment scar deposition through fibroblast mechanotransduction. In idiopathic pulmonary fibrosis, a fatal form of progressive lung fibrosis, previous work has shown that loss of Thy-1 (CD90) expression in fibroblasts correlates with regions of active fibrogenesis, thus representing a pathologically relevant fibroblast subpopulation. We now show that Thy-1 is a regulator of fibroblast rigidity sensing. Thy-1 physically couples to inactive αvβ3 integrins via its RGD-like motif, altering baseline integrin avidity to ECM ligands and also facilitating preadhesion clustering of integrin and membrane rafts via Thy-1's glycophosphatidylinositol tether. Disruption of Thy-1-αvβ3 coupling altered recruitment of Src family kinases to adhesion complexes and impaired mechanosensitive, force-induced Rho signaling, and rigidity sensing. Loss of Thy-1 was sufficient to induce myofibroblast differentiation in soft ECMs and may represent a physiological mechanism important in wound healing and fibrosis.

Lentiviral vector-mediated transduction of goat undifferentiated spermatogonia

Recent studies show that spermatogonial stem cells (SSCs) are able to colonize and form mature spermatozoa following transplantation into germ cell depleted testes of recipient males. Therefore, efficient ways for enrichment and gene transfer into SSCs provides a powerful tool for production of transgenic animals. In order to adapt the technique to goats, three issues were addressed: (i) enrichment of the undifferentiated spermatogonia including SSCs using magnetic activated cell sorting (MACS), (ii) lentiviral vector-mediated transduction of an enhanced green fluorescent protein (EGFP) transgene into enriched cells, and (iii) transplantation of transduced undifferentiated spermatogonia into the germ cell depleted testes of immune-suppressed mice to assess for migration and colony formation ability. Enriched cells were transduced by lentiviral vectors and subsequently analyzed for expression of THY1, PLZF, VASA, UCHL1 and BCL6B genes. Cells were also analyzed for GFP and PLZF by flow cytometry. Enriched transduced cells were transplanted into germ cell depleted mice testis. Quantitative analysis of transcripts revealed that MACS-enrichment significantly increased the expression of SSC-characteristic genes THY1, PLZF, VASA, UCHL1 and BCL6B compared to non-enriched population (P≤0.05). EGFP transduction did not affect the expression levels of SSC-characteristic genes. Flow cytometry revealed that 72% of transduced-enriched cells were positive for EGFP. Finally, transduced-enriched goat SSCs could colonize within the cells into the seminiferous tubules of germ cell depleted recipient mice at higher frequency than non-enriched cells. The results indicated that enrichment of goat undifferentiated spermatogonia by magnetic-activated cell sorting for THY1 antibody combined with lentiviral vector-mediated transduction has the potential to be used for production of transgenic goats.

TGF-β1 epigenetically modifies Thy-1 expression in primary lung fibroblasts.

Idiopathic pulmonary fibrosis is a progressive lung disease that increases in incidence with age. We identified a profibrotic lung phenotype in aging mice characterized by an increase in the number of fibroblasts lacking the expression of thymocyte differentiation antigen 1 (Thy-1) and an increase in transforming growth factor (TGF)-β1 expression. It has been shown that Thy-1 expression can be epigenetically modified. Lung fibroblasts (PLFs) were treated with TGF-β1 ± DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-AZA) and analyzed for Thy-1 gene and protein expression, DNMT protein expression, and activity. α-Smooth muscle actin (α-SMA) and collagen type 1 (Col1A1) gene and protein expression was assessed. PLFs were transfected with DNMT1 silencing RNA ± TGF-β1. TGF-β1 inhibited Thy-1 gene and protein expression in PLFs, and cotreatment with 5-AZA ameliorated this effect and appeared to inhibit DNMT1 activation. TGF-β1 induced Thy-1 promoter methylation as assessed by quantitative methyl PCR. Treatment with 5-AZA attenuated TGF-β1-induced Col1A1 gene and protein expression and α-SMA gene expression (but not α-SMA protein expression). Inhibiting DNMT1 with silencing RNA attenuated TGF-β1-induced DNMT activity and its downstream suppression of Thy-1 mRNA and protein expression as well as inhibited α-SMA mRNA and Col1A1 mRNA and protein expression, and showed a decreased trend in Thy-1 promoter methylation. Immunofluorescence for α-SMA suggested that 5-AZA inhibited stress fiber formation. These findings suggest that TGF-β1 epigenetically regulates lung fibroblast phenotype through methylation of the Thy-1 promoter. Targeted inhibition of DNMT in the right clinical context might prevent fibroblast to myofibroblast transdifferentiation and collagen deposition, which in turn could prevent fibrogenesis in the lung and other organs.