MicroRNA-7 promotes neural differentiation of trabecular meshwork mesenchymal stem cell on nanofibrous scaffold

The purpose of this study was to investigate miR-7 overexpression effects on neural differentiation of mesenchymal stem cells (MSCs) on both two-dimensional (2D) and three-dimensional (3D) culture systems. We upregulated miR-7 through lentiviral vector in trabecular meshwork MSCs (TMMSCs) and polymers of poly l -lactic acid/polycaprolactone fibrous scaffold were fabricated by electrospinning and characterized using scanning electron microscopy (SEM) and Fourier transform infrared (FTIR). Neural markers expression was evaluated through quantitative-polymerase chain reaction (q-PCR) and immunostaining. The results showed that the high percentage of cell transduction (84.9%) and miR-7 expression (folds: 677.93 and 556.4) was detected in TMMSCs-miR-7(+). SEM and FTIR established the fabrication of the hybrid scaffold. q-PCR analysis showed that on days 14 and 21 of transduction, the expression level of Nestin and glial fibrillary acidic protein (GFAP) genes were significantly higher in the scaffold (3D) compared with tissue culture polystyrene (2D) culture. The expression of microtubule-associated protein-2 (MAP-2) and GFAP genes in TMMSCs-miR-7(+) cells were significantly higher than those miR-7(−) cells after 21 days of cell culture. Also, MAP-2 and Nestin proteins were detected in TMMSCs-miR-7(+) cells. Our results demonstrate that miR-7 is involved in neural differentiation of TMMSCs and scaffold can improve differentiate into glial and neural progenitor cells. These findings provided some information for future use of microRNAs and scaffold in tissue engineering and cell therapy for neurological diseases.     

Possible role of differentially expressing novel protein markers (ligatin and fibulin‐7) in human aqueous humor and trabecular meshwork tissue in glaucoma progression

The pathological mechanism underlying glaucoma has always been a complex aspect of this permanently blinding disease but proteomic studies have been helpful in elucidating it to a great extent in several studies. This study was designed to evaluate the expression and to get an idea about the function of two novel markers (ligatin and fibulin‐7) identified in human aqueous humor (hAH) in relation to glaucomatous progression. A significant increase in the protein content of glaucomatous hAH compared to that of non‐glaucomatous controls (NG‐Ctrls) was observed. Ligatin, fibulin‐7, and its proteolysis were revealed in hAH of primary open angle glaucoma (POAG), primary angle closure glaucoma (PACG) and NG‐Ctrls. Quantification confirmed no significant difference in expression of ligatin, whereas fibulin‐7 was significantly (P < 0.05) low in hAH of PACG in comparison to NG‐Ctrls and POAG. Importantly the immunohistochemical assay for both indicated their possible involvement in the maintenance of the appropriate structure of TM in vivo. Since oxidative stress is a major contributor to glaucomatous pathogenesis, in vitro analysis of nuclear and cytoplasmic fractions indicated intracellular changes in localization and expression of ligatin upon oxidative insult of human trabecular meshwork (TM) cells. While no such changes were found for fibulin‐7 expression. This was also corroborated with the immunocytochemical assay. Though a study with a small sample size, this is the first report which confirms the presence of ligatin and fibulin‐7 in hAH, quantified their differential expression, and indicated the possibility of their involvement in the maintenance of the TM structure.     

The regulation of connective tissue growth factor expression influences the viability of human trabecular meshwork cells

Connective tissue growth factor (CTGF) induces extracellular matrix (ECM) synthesis and contractility in human trabecular meshwork (HTM) cells. Both processes are involved in the pathogenesis of primary open‐angle glaucoma. To date, little is known about regulation and function of CTGF expression in the trabecular meshwork (TM). Therefore, we analysed the effects of different aqueous humour proteins and stressors on CTGF expression in HTM cells. HTM cells from three different donors were treated with endothelin‐1, insulin‐like growth factor (IGF)‐1, angiotensin‐II, H₂O₂ and heat shock and were analysed by immunohistochemistry, real‐time RT‐PCR and Western blotting. Viability after H₂O₂ treatment was measured in CTGF silenced HTM‐N cells and their controls. Latrunculin A reduced expression of CTGF by about 50% compared to untreated HTM cells, whereas endothelin‐1, IGF‐1, angiotensin‐II, heat shock and oxidative stress led to a significant increase. Silencing of CTGF resulted in a delayed expression of αB‐crystallin and in reduced cell viability in comparison to the controls after oxidative stress. Conversely, CTGF treatment led to a higher cell viability rate after H₂O₂ treatment. CTGF expression is induced by factors that have been linked to glaucoma. An increased level of CTGF appears to protect TM cells against damage induced by stress. The beneficial effect of CTGF for viability of TM cells is likely associated with the effects on increased ECM synthesis and higher contractility of the TM, thereby contributing to reduced aqueous humour outflow facility causing increased intraocular pressure.     

Cytokine profile and cytoskeletal changes after herpes simplex virus type 1 infection in human trabecular meshwork cells

Uveitis caused by herpes simplex virus (HSV)-1 is characterized by increased intraocular pressure (IOP) in the presence of anterior chamber inflammation. Despite their clinical significance, the pathogenic changes associated with HSV-1 infection in trabecular meshwork (TM) cells, the key cell type regulating IOP, have not been completely elucidated. In this study, cytokine array analyses showed a significant stepwise increase in monocyte chemoattractant protein (MCP)-1 expression upon HSV-1 infection in TM cells (p < 0.05). HSV-1 infection led to downregulation of fibrogenic molecules (fibronectin, α-smooth muscle actin, connective tissue growth factor and TGF-β1). Notably, HSV-1 infection caused a significant increase in actin stress fibres, with a twofold increase in active RhoA, which was enhanced by treatment with TGF-β1 and inhibited by treatment with the Rho-kinase inhibitor, Y-27632. TM cells treated with MCP-1 exhibited a dose-dependent increase in actin stress fibres compared to untreated TM cells. Our study suggests that HSV-1 infection in TM cells increases cell contractile activity rather than fibrotic changes in the extracellular matrix (ECM) components. Taken together, these observations demonstrate the enhanced expression of MCP-1 and TM cell contractile activity upon HSV-1 infection and events with potential implications for the pathobiology of abrupt IOP elevation in HSV-1 anterior uveitis.     

Role of vasorin,an anti‐apoptotic,anti‐TGF‐β and hypoxia‐induced glycoprotein in the trabecular meshwork cells and glaucoma

Glaucoma, one of the leading causes of irreversible blindness, is commonly associated with elevated intraocular pressure due to impaired aqueous humour (AH) drainage through the trabecular meshwork. The aetiological mechanisms contributing to impaired AH outflow, however, are poorly understood. Here, we identified the secreted form of vasorin, a transmembrane glycoprotein, as a common constituent of human AH by mass spectrometry and immunoblotting analysis. ELISA assay revealed a significant but marginal decrease in vasorin levels in the AH of primary open‐angle glaucoma patients compared to non‐glaucoma cataract patients. Human trabecular meshwork (HTM) cells were confirmed to express vasorin, which has been shown to possess anti‐apoptotic and anti‐TGF‐β activities. Treatment of HTM cells with vasorin induced actin stress fibres and focal adhesions and suppressed TGF‐β2‐induced SMAD2/3 activation in HTM cells. Additionally, cobalt chloride‐induced hypoxia stimulated a robust elevation in vasorin expression, and vasorin suppressed TNF‐α‐induced cell death in HTM cells. Taken together, these findings reveal the importance of vasorin in maintenance of cell survival, inhibition of TGF‐β induced biological responses in TM cells, and the decreasing trend in vasorin levels in the AH of glaucoma patients suggests a plausible role for vasorin in the pathobiology of ocular hypertension and glaucoma.     

Establishment and characterization of an in vitro 3D ovarian cancer model for drug screening assays

The acquired drug chemoresistance represents the main challenge of the ovarian cancer treatment. In addition, the absence of an adequate in vitro model able to reproduce the native tumor environment can contribute to the poor success rate of pre-clinical studies of new compounds. Three-dimensional (3D) culture models have been recently used for drug screening purposes due to their ability to reproduce the main characteristics of in vivo solid tumors. Here we describe the establishment and characterization of 3D ovarian cancer spheroids using different adenocarcinoma tumor cell lines (SKOV-3 and OVCAR-3 cells) in two different 3D scaffold-free methods: forced-floating in ultra-low attachment (ULA) plates and hanging drop (HD). Spheroids were evaluated in both 3D cultures in order to establish the best condition to perform the drug response analysis with Paclitaxel, a common drug used to treat ovarian cancer. SKOV-3 and OVCAR-3 spheroids with the desired characteristics (roundness close to 1.0 and diameter in the 200–500 μm range) were obtained using both methods after addition of the methylcellulose (MC) in the culture medium (0.25% and 0.5%, w/v). We also observed the presence of microvilli on the surface of the spheroids, higher presence of apoptotic cells and higher drug resistance, when compared with 2D cultures. The 3D cultures obtained seem to provide more reliable results in terms of drug response than those provided by 2D monolayer culture. The forced floating method was considered more suitable and straightforward to generate ovarian cancer spheroids for drug screening/cytotoxicity assays.     

MTOR-independent induction of autophagy in trabecular meshwork cells subjected to biaxial stretch

The trabecular meshwork (TM) is part of a complex tissue that controls the exit of aqueous humor from the anterior chamber of the eye, and therefore helps maintaining intraocular pressure (IOP). Because of variations in IOP with changing pressure gradients and fluid movement, the TM and its contained cells undergo morphological deformations, resulting in distention and stretching. It is therefore essential for TM cells to continuously detect and respond to these mechanical forces and adapt their physiology to maintain proper cellular function and protect against mechanical injury. Here we demonstrate the activation of autophagy, a pro-survival pathway responsible for the degradation of long-lived proteins and organelles, in TM cells when subjected to biaxial static stretch (20% elongation), as well as in high-pressure perfused eyes (30 mm Hg). Morphological and biochemical markers for autophagy found in the stretched cells include elevated LC3-II levels, increased autophagic flux, and the presence of autophagic figures in electron micrographs. Furthermore, our results indicate that the stretch-induced autophagy in TM cells occurs in an MTOR- and BAG3-independent manner. We hypothesize that activation of autophagy is part of the physiological response that allows TM cells to cope and adapt to mechanical forces.     

In vitro 3D human small intestinal villous model for drug permeability determination

We present a novel method for testing drug permeability that features human cells cultured on hydrogel scaffolds made to accurately replicate the shape and size of human small intestinal villi. We compared villous scaffolds to more conventional 2D cultures in paracellular drug absorption and cell growth experiments. Our results suggest that 3D villous platforms facilitate cellular differentiation and absorption more similar to mammalian intestines than can be achieved using conventional culture. To the best of our knowledge, this is the first accurate 3D villus model offering a well-controlled microenvironment that has strong physiological relevance to the in vivo system.     

UVB-induced production of 1,25-dihydroxyvitamin D3 and vitamin D activity in human keratinocytes pretreated with a sterol delta7-reductase inhibitor

The skin fulfills an important role in the vitamin D photo-endocrine system. Epidermis is not only the site of vitamin D3 photoproduction. In addition, epidermal keratinocytes contain the vitamin D receptor (VDR) and possess 25-hydroxylase and 1alpha-hydroxylase activity indicating that all components of the vitamin D system are present. We investigated whether these components cooperate in inducing vitamin D activity upon treatment with physiological UVB doses. Upon irradiation, 24-hydroxylase mRNA was induced in keratinocytes pretreated with a sterol Delta7-reductase inhibitor (BM15766) whereby the 7-dehydrocholesterol content increased by 300-fold. Transfection experiments with a vitamin D response element containing construct confirmed VDR-dependent gene activation. Furthermore, the UVB-dependent induction of 24-hydroxylase was blocked by the cytochrome-P450 inhibitor ketoconazole. The 24-hydroxylase inducing photoproduct was transferable to unirradiated keratinocytes by medium and cellular homogenates of UVB-irradiated, BM15766-pretreated cells and was identified as 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] by high-performance liquid chromatography with tandem mass spectrometric detection. Addition of vitamin D binding protein blunted UVB-induced 24-hydroxylase suggesting the possibility of a paracrine or autocrine role for 1,25(OH)2D3. In conclusion, epidermal keratinocytes can produce vitamin D3, convert it to 1,25(OH)2D3 and respond to it upon UVB irradiation in the absence of exogenous 7-dehydrocholesterol and therefore contain a unique and complete photo-endocrine vitamin D system.     

The vitamin D receptor-mediated activation of phosphatidylinositol 3-kinase (PI3Kalpha) plays a role in the 1alpha,25-dihydroxyvitamin D3-stimulated increase in steroid sulphatase activity in myeloid leukaemic cell lines

In this article we show that 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) stimulates the activity of the class IA phosphatidylinositol 3-kinase PI3Kalpha and its downstream target Akt in HL60, U937 and THP-1 myeloid leukaemic cell lines. Furthermore, we show that the classical nuclear vitamin D receptor (VDR(nuc)) is involved in this activation of the PI3K/Akt signalling in these cell lines. We have previously shown that the activity of steroid sulphatase is stimulated in HL60, U937 and THP-1 myeloid leukaemic cell lines by 1alpha,25(OH)(2)D(3) (Hughes et al., [2001] Biochem J 355:361-371; Hughes et al., [2005] J Cell Biochem 94:1175-1189; Hughes and Brown [2006] J Cell Biochem 98:590-617). In this article we show that the 1alpha,25(OH)(2)D(3)-stimulated increase in signalling via the PI3K/Akt pathway plays a role in the increase in steroid sulphatase activity in the HL60 U937 and THP-1 cell lines. We used a variety of pharmacological and biochemical approaches to show that activation of PI3Kalpha mediates the 1alpha,25(OH)(2)D(3)-stimulated increase in steroid sulphatase activity in myeloid leukaemic cells. We also show that the PI3K/Akt dependent activation of NF-kappaB plays a role in the 1alpha,25(OH)(2)D(3)-stimulated increase in steroid sulphatase activity in myeloid leukaemic cells.     

Identification of predictive genetic signatures of Cytarabine responsiveness using a 3D acute myeloid leukaemia model

This study reports the establishment of a bone marrow mononuclear cell (BMMC) 3D culture model and the application of this model to define sensitivity and resistance biomarkers of acute myeloid leukaemia (AML) patient bone marrow samples in response to Cytarabine (Ara‐C) treatment. By mimicking physiological bone marrow microenvironment, the growth conditions were optimized by using frozen BMMCs derived from healthy donors. Healthy BMMCs are capable of differentiating into major hematopoietic lineages and various types of stromal cells in this platform. Cryopreserved BMMC samples from 49 AML patients were characterized for ex vivo growth and sensitivity to Ara‐C. RNA sequencing was performed for 3D and 2D cultures to determine differential gene expression patterns. Specific genetic mutations and/or gene expression signatures associated with the ability of the ex vivo expansion and response to Ara‐C were elucidated by whole‐exome and RNA sequencing. Data analysis identified unique gene expression signatures and novel genetic mutations associated with sensitivity to Ara‐C treatment of proliferating AML specimens and can be used as predictive therapeutic biomarkers to determine the optimal treatment regimens. Furthermore, these data demonstrate the translational value of this ex vivo platform which should be widely applicable to evaluate other therapies in AML.     

Oxygen tension differentially influences osteogenic differentiation of human adipose stem cells in 2D and 3D cultures

Skeletal defects commonly suffer from poor oxygen microenvironments resulting from compromised vascularization associated with injury or disease. Adipose stem cells (ASCs) represent a promising cell population for stimulating skeletal repair by differentiating toward the osteogenic lineage or by secreting trophic factors. However, the osteogenic or trophic response of ASCs to reduced oxygen microenvironments is poorly understood. Moreover, a direct comparison between 2D and 3D response of ASCs to hypoxia is lacking. Thus, we characterized the osteogenic and angiogenic potential of human ASCs under hypoxic (1%), normoxic (5%), and atmospheric (21%) oxygen tensions in both 2D and 3D over 4 weeks in culture. We detected greatest alkaline phosphatase activity and extracellular calcium deposition in cells cultured in both 2D and 3D under 21% oxygen, and reductions in enzyme activity corresponded to reductions in oxygen tension. ASCs cultured in 1% oxygen secreted more vascular endothelial growth factor (VEGF) over the 4‐week period than cells cultured in other conditions, with cells cultured in 2D secreting VEGF in a more sustained manner than those in 3D. Expression of osteogenic markers revealed temporal changes under different oxygen conditions with peak expression occurring earlier in 3D. In addition, the increase of most osteogenic markers was significantly higher in 2D compared to 3D cultures at 1% and 5% oxygen. These results suggest that oxygen, in conjunction with dimensionality, affects the timing of the differentiation program in ASCs. These findings offer new insights for the use of ASCs in bone repair while emphasizing the importance of the culture microenvironment. J. Cell. Biochem. 110: 87–96, 2010. © 2010 Wiley‐Liss, Inc.     

Increased strength in the Col-Tgel induces apoptosis in the human dental pulp stem cells: 3D culturing of human dental pulp stem cells at different strengths of collagen

Human dental pulp stem cells (HDPSCs) have great potential to be used in regenerative medicine. To use these stem cells effectively for this purpose, they should be grown in a 3D cell culture that mimics their natural niches instead of a 2D conventional cell culture. The aim of this study was to grow the HDPSCs in the 3D cell culture created by Transglutaminase-crosslinked collagen hydrogels (Col-Tgel) in two different strengths to find a suitable 3D cell culture environment for these stem cells. Two stiffness of the 3D Col-Tgel were used to grow the HDPSCs: soft and medium matrix with strength of 0.9–1.5 kPa and 14–20 kPa, respectively. HDPSCs express markers similar to MSCs, therefore seven such markers were analyzed in the HDPSCs during their growth in the 2D and in the 3D soft and medium Col-Tgel. The CD105 and CD90 markers were significantly (p < 0.05) downregulated in HDPSCs cultured in both 3D cell culture conditions compared with HDPSCs in 2D cell culture. Furthermore, CD34 marker, a negative marker, expressed by a few cells in HDPSCs culture was upregulated (p < 0.05) in HDPSCs cultured in medium 3D Col-Tgel, indicating cells that expressing the marker grow better in medium 3D Col-Tgel. The apoptosis results revealed that HDPSCs in medium 3D Col-Tgel had the least number of live cells and a significantly (p < 0.05) higher early apoptosis rate compared to HDPSCs in 2D and 3D Col-Tgel medium. MTT analysis also showed a significant difference among the three cell culture conditions. We conclude that HDPSCs cultured on 3D soft Col-Tgel showed better proliferation than cells cultured in 3D medium gel. These results demonstrate that the ideal environment to grow HDPSCs in 3D is the soft Col-Tgel not medium Col-Tgel.