Differential regulation of inducible nitric oxide synthase and cyclooxygenase-2 expression by superoxide dismutase in lipopolysaccharide stimulated RAW 264.7 cells

Inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) have been known to be involved in various pathophysiological processes such as inflammation. This study was performed to determine the regulatory function of superoxide dismutase (SOD) on the LPS-induced expression of iNOS, and COX-2 in RAW 264.7 cells. When a cell-permeable SOD, Tat-SOD, was added to the culture medium of RAW 264.7 cells, it rapidly entered the cells in a dose-dependent manner. Treatment of RAW 264.7 cells with Tat-SOD led to decrease in LPS-induced ROS generation. Pretreatment with Tat-SOD significantly inhibited LPS-induced expression of iNOS and NO production but had no effect on the expression of COX-2 and PGE₂ production in RAW 264.7 cells. Tat-SOD inhibited LPS-induced NF-κB DNA binding activity, IκBα degradation and activation of MAP kinases. These data suggest that SOD differentially regulate expression of iNOS and COX-2 in LPS-stimulated RAW 264.7 cells.     

Human Elastin‐Based Recombinant Biopolymers Improve Mesenchymal Stem Cell Differentiation

Elastin‐based polypeptides are a class of smart biopolymers representing an important model in the design of biomaterials. The combination of biomimetic materials with cells that have great plasticity provides a promising strategy for the realization of highly engineered cell‐based constructs for regenerative medicine and tissue repair applications. Two recombinant biopolymers inspired by human elastin are assessed as coating agents to prepare biomimetic surfaces for cell culture. These substrates are assayed for hBM MSC culture. The coated surfaces are also characterized with AFM to evaluate the topographical features of the deposited biopolymers. The results suggest that the elastin‐derived biomimetic surfaces play a stimulatory role on osteogenic differentiation of MSCs.

     

Effect of Composition of Lung Biomimetic Niche on the Mesenchymal Stem Cell Differentiation toward Alveolar Type II Pneumocytes

Pulmonary niche dynamically orchestrates the signals, such as proliferation or differentiation of mesenchymal stem cells (MSCs), which allows inducing tissue repair. Lung niche includes extracellular matrix (ECM), comprising hyaluronic acid (HA) and collagen (COLL), and several types of MSCs. Impaired ECM, in lung pathologies, makes the promising therapies based on MSCs ineffective, as it results in a reduced attachment and homing of MSCs, precluding their differentiation and viability. To overcome this problem, in this study a pulmonary biomimetic niche based on HA and COLL hydrogel is developed, with the specific aim to elucidate the role of COLL and HA/COLL semi-interpenetrating polymer networks (SIPNs) in directing the differentiation of MSCs into Alveolar Type II (ATII) cells. The effect of low (L), medium (M), and high (H) molecular weight (MW) HA is investigated, both like structural component of the SIPNs hydrogel and like trophic factor in cell culture media solution. HA in the culture media significantly improves surfactant protein (SP)-C expression (≈2 ng mL⁻¹), without showing difference in the MW tested, compared to control only (≈1 ng mL⁻¹). Furthermore, LMWHA/COLL hydrogel promotes the SPC expression (approximately two times) compared to COLL, MMWHA/COLL, and HMWHA/COLL hydrogels.     

Expression patterns of betaig-h3 in chondrocyte differentiation during endochondral ossification

betaig-h3 is a TGF-beta-induced extracellular matrix protein which is expressed in many tissues including bones and cartilages. In previous reports, we showed that betaig-h3 mediates cell adhesion and migration and, especially in bones, negatively regulates the mineralization in the end stage of endochondral ossification. Here, to elucidate the expression pattern and role of betaig-h3 in chondrocyte differentiation, ATDC5 chondrocytes and embryonic and postnatal mice were used for in vitro differentiation studies and in vivo studies, respectively. betaig-h3 was strongly induced by the treatment of TGF-beta1 and the expression level of betaig-h3 mRNA and protein were highly expressed in the early stages of differentiation but decreased in the late stages in ATDC5. Furthermore, the patterns of TGF-beta1, -beta2, and -beta3 mRNA expression were concurrent with betaig-h3 in ATDC5. betaig-h3 was deeply stained in perichondrium (PC), periosteum (PO), and prehypertrophic chondrocytes (PH) through the entire period of endochondral ossification in mice. betaig-h3 was mainly expressed in PC and PH at embryonic days and obviously in PH in postnatal days. These results suggest that betaig-h3 may play a critical role as a regulator of chondrogenic differentiation in endochondral ossification.     

Modulation of cartilage differentiation by melanoma inhibiting activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP)

Melanoma inhibiting activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP) is a small soluble protein secreted from malignant melanoma cells and from chondrocytes. Recently, we revealed that MIA/CD-RAP can modulate bone morphogenetic protein (BMP)2-induced osteogenic differentiation into a chondrogenic direction. In the current study we aimed to find the molecular details of this MIA/CD-RAP function. Direct influence of MIA on BMP2 by protein-protein-interaction or modulating SMAD signaling was ruled out experimentally. Instead, we revealed inhibition of ERK signaling by MIA/CD-RAP. This inhibition is regulated via binding of MIA/CD-RAP to integrin α5 and abolishing its activity. Active ERK signaling is known to block chondrogenic differentiation and we revealed induction of aggrecan expression in chondrocytes by treatment with MIA/CD-RAP or PD098059, an ERK inhibitor. In in vivo models we could support the role of MIA/CD-RAP in influencing osteogenic differentiation negatively. Further, MIA/CD-RAP-deficient mice revealed an enhanced calcified cartilage layer of the articular cartilage of the knee joint and disordered arrangement of chondrocytes. Taken together, our data indicate that MIA/CD-RAP stabilizes cartilage differentiation and inhibits differentiation into bone potentially by regulating signaling processes during differentiation.     

Shank 2 expression coincides with neuronal differentiation in the developing retina

The retinal activity for vision requires a precise synaptic connectivity. Shank proteins at postsynaptic sites of excitatory synapses play roles in signal transmission into the postsynaptic neuron. However, the correlation of Shank 2 expression with neuronal differentiation in the developing retina remains to be elucidated regardless of previous evidences of Shank 2 expression in retina. Herein, we demonstrated that with progression of development, Shank 2 is initially detected in the inner plexiform layer at P2, and then intensively detected in inner plexiform layer, outer plexiform layer, and ganglion cell layer at P14, which was closely colocalized to the neurofilament expression. Shank 2 was, however, not colocalized with glial fibrillary acidic protein. Shank 2 expression was increased in the differentiated retinoblastoma cells, which was mediated by ERK 1/2 activation. Moreover, Shank 2 expression was colocalized with neurofilament at the dendritic region of cells. In conclusion, our data suggests that Shank 2 is expressed in the neurons of the developing retina and could play a critical role in the neuronal differentiation of the developing retina.     

Differentiation of human labia minora dermis-derived fibroblasts into insulin-producing cells

Recent evidence has suggested that human skin fibroblasts may represent a novel source of therapeutic stem cells. In this study, we report a 3-stage method to induce the differentiation of skin fibroblasts into insulin- producing cells (IPCs). In stage 1, we establish the isolation, expansion and characterization of mesenchymal stem cells from human labia minora dermis- derived fibroblasts (hLMDFs) (stage 1: MSC expansion). hLMDFs express the typical mesenchymal stem cell marker proteins and can differentiate into adipocytes, osteoblasts, chondrocytes or muscle cells. In stage 2, DMEM/F12 serum-free medium with ITS mix (insulin, transferrin, and selenite) is used to induce differentiation of hLMDFs into endoderm-like cells, as determined by the expression of the endoderm markers Sox17, Foxa2, and PDX1 (stage 2: mesenchymal-endoderm transition). In stage 3, cells in the mesenchymal- endoderm transition stage are treated with nicotinamide in order to further differentiate into self-assembled, 3-dimensional islet cell-like clusters that express multiple genes related to pancreatic β-cell development and function (stage 3: IPC). We also found that the transplantation of IPCs can normalize blood glucose levels and rescue glucose homeostasis in streptozotocin- induced diabetic mice. These results indicate that hLMDFs have the capacity to differentiate into functionally competent IPCs and represent a potential cell-based treatment for diabetes mellitus.     

Improved efficacy of bio‐mineralization of human mesenchymal stem cells on modified PLLA nanofibers coated with bioactive materials via enhanced expression of integrin α2β1

Current therapeutic interventions in bone defects are mainly focused on finding the best bioactive materials for inducing bone regeneration via activating the related intracellular signaling pathways. Integrins are trans‐membrane receptors that facilitate cell‐extracellular matrix (ECM) interactions and activate signal transduction. To develop a suitable platform for supporting human bone marrow mesenchymal stem cells (hBM‐MSCs) differentiation into bone tissue, electrospun poly L‐lactide (PLLA) nanofiber scaffolds were coated with nano‐hydroxyapatite (PLLA/nHa group), gelatin nanoparticles (PLLA/Gel group), and nHa/Gel nanoparticles (PLLA/nHa/Gel group) and their impacts on cell proliferation, expression of osteoblastic biomarkers, and bone differentiation were examined and compared. MTT data showed that proliferation of hBM‐MSCs on PLLA/nHa/Gel scaffolds was significantly higher than other groups (P < .05). Alkaline phosphatase activity was also more increased in hBM‐MSCs cultured under osteogenic media on PLLA/nHa/Gel scaffolds compared to others. Gene expression evaluation confirmed up‐regulation of integrin α2β1 as well as the osteogenic genes BGLAP, COL1A1, and RUNX2. Following use of integrin α2β1 blocker antibody, the protein level of integrin α2β1 in cells seeded on PLLA/nHa/Gel scaffolds was decreased compared to control, which confirmed that most of the integrin receptors were bound to gelatin molecules on scaffolds and could activate the integrin α2β1/ERK axis. Collectively, PLLA/nHa/Gel scaffold is a suitable platform for hBM‐MSCs adhesion, proliferation, and osteogenic differentiation in less time via activating integrin α2β1/ERK axis, and thus it might be applicable in bone tissue engineering.     

Mesenchymal stem cells promote proliferation of endogenous neural stem cells and survival of newborn cells in a rat stroke model

Mesenchymal stem cells (MSCs) secrete bioactive factors that exert diverse responses in vivo. In the present study, we explored mechanism how MSCs may lead to higher functional recovery in the animal stroke model. Bone marrow-derived MSCs were transplanted into the brain parenchyma 3 days after induction of stroke by occluding middle cerebral artery for 2 h. Stoke induced proliferation of resident neural stem cells in subventricular zone. However, most of new born cells underwent cell death and had a limited impact on functional recovery after stroke. Transplantation of MSCs enhanced proliferation of endogenous neural stem cells while suppressing the cell death of newly generated cells. Thereby, newborn cells migrated toward ischemic territory and differentiated in ischemic boundaries into doublecortin+ neuroblasts at higher rates in animals with MSCs compared to control group. The present study indicates that therapeutic effects of MSCs are at least partly ascribed to dual functions of MSCs by enhancing endogenous neurogenesis and protecting newborn cells from deleterious environment. The results reinforce the prospects of clinical application using MSCs in the treatment of neurological disorders.     

TCP复合BMP-2诱导hMSCs成骨分化的协调机制研究

目的探讨TCP复合BMP-2诱导h MSCs成骨分化的协调机制。方法分别制备TCP支架及TCP复合BMP-2支架材料,自3名志愿者身上分离h MSCs进行传代扩增,取第3代细胞接种备好的支架材料,在复合后的第1、3、7天测量ALP的活性并进行比较。结果在复合后第1天,两组间ALP活性值比较无明显差异(P0.05);在复合后第3、7天两组间ALP活性值比较差异显著(P0.05);TCP与细胞培养组1、3、7天的ALP活性值差异不显著,无统计学意义(P0.05);TCP复合BMP-2与细胞培养组的1、3、7天的ALP活性值随着培养时间的延长,逐渐升高。结论 BMP-2能协调TCP促进h MSCs的分化效应,基因治疗可能为骨骼疾病提供一种新的治疗途径。     

AMP kinase/cyclooxygenase-2 pathway regulates proliferation and apoptosis of cancer cells treated with quercetin

AMPK (AMP-activated protein kinase) is highly conserved in eukaryotes, where it functions primarily as a sensor of cellular energy status. Recent studies indicate that AMPK activation strongly suppresses cell proliferation in non-malignant cells as well as in tumor cells. In this study, quercetin activated AMPK in MCF breast cancer cell lines and HT-29 colon cancer cells, and this activation of AMPK seemed to be closely related to a decrease in COX-2 expression. The application of a COX-2 inhibitor or cox-2^-/- cells supported the idea that AMPK is an upstream signal of COX-2, and is required for the anti-proliferatory and pro-apoptotic effects of quercetin. The suppressive or growth inhibitory effects of quercetin on COX-2 were abolished by treating cancer cells with an AMPK inhibitor Compound C. These results suggest that AMPK is crucial to the anti-cancer effect of quercetin and that the AMPK-COX-2 signaling pathway is important in quercetin-mediated cancer control.     

Effect of sildenafil citrate on interleukin-1beta-induced nitric oxide synthesis and iNOS expression in SW982 cells

The purpose of this study was to identify the effect of sildenafil citrate on IL-1β-induced nitric oxide (NO) synthesis and iNOS expression in human synovial sarcoma SW982 cells. IL-1β stimulated the cells to generate NO in both dose- and time-dependent manners. The IL-1β-induced NO synthesis was inhibited by guanylate cyclase (GC) inhibitor, LY83583. When the cells were treated with 8-bromo-cGMP, a hydrolyzable analog of cGMP, NO synthesis was increased upto 5-fold without IL-1β treatment suggesting that cGMP is an essential component for increasing the NO synthesis. Synoviocytes and chondrocytes contain strong cGMP phosphodiesterase (PDE) activity, which has biochemical features of PDE5. When SW982 cells were pretreated with sildenafil citrate (Viagra), a PDE5 specific inhibitor, sildenafil citrate significantly inhibited IL-1β-induced NO synthesis and iNOS expressions. From this result, we noticed that PDE5 activity is required for IL-1β-induced NO synthesis and iNOS expressions in human synovial sarcoma cells, and sildenafil citrate may be able to suppress an inflammatory reaction of synovium through inhibition of NO synthesis and iNOS expression by cytokines.     

Spectroscopic Behaviour of Copper(II) Complexes Containing 2-Hydroxyphenones

Theoretical investigations by density functional theory (DFT) and time-dependent DFT (TD-DFT) methods shed light on how the type of ligand or attached groups influence the electronic structure, absorption spectrum, electron excitation, and intramolecular and interfacial electron transfer of the Cu(II) complexes under study. The findings provide new insight into the designing and screening of high-performance dyes for dye-sensitized solar cells (DSSCs).     

Effects of adenovirus-mediated bFGF, IL-1Ra and IGF-1 gene transfer on human osteoarthritic chondrocytes and osteoarthritis in rabbits

The study investigated the effects of adenovirus-mediated gene transfection of basic fibroblast growth factor (bFGF), bFGF combined with interleukin-1 receptor antagonist protein (IL-Ra) and/or insulin-like growth factor-1 (IGF-1) both in human osteoarthritis (OA) chondrocytes and rabbits OA model. Human OA chondrocytes were delivered by adenovirus-mediated bFGF, IL-Ra and IGF-1 vectors, respectively. Chondrocyte proliferation, glycosaminoglycan (GAG) content, expression of type II collagen, ADAMTS-5, MMP-13, MMP-3 and TIMP-1 were determined. Rabbit OA model was induced by anterior cruciate ligament transaction (ACLT) in knees. Adenoviral vectors encoding human bFGF, IL-Ra and IGF-1 were injected intraarticularly into the knee joints after ACLT. The effects of adenovirus- mediated gene transfection on rabbit OA were evaluated. In vitro, the transfected genes were expressed in cell supernatant of human OA chondrocytes. AdbFGF group significantly promoted chondrocyte proliferation, and increased GAG and type II collagen synthesis than in the OA group. As two or three genes were transfected in different combinations, there was significant enhancement on the GAG content, type II collagen synthesis, and TIMP-1 levels, while ADAMTS-5, MMP-13, and MMP-3 levels were reduced. In vivo, the transfected genes were expressed in synovial fluid of rabbits. Intraarticular delivery of bFGF enhanced the expression of type II collagen in cartilage and decreased cartilage Mankin score compared with the OA control group (P = 0.047; P < 0.01, respectively). Multiple-gene transfection in different combinations showed better results than bFGF transfection alone. This study suggests that bFGF gene transfection is effective in treating experimental OA. Multiple gene transfection has better biologic effects on OA.     

In vitro migration capacity of human adipose tissue-derived mesenchymal stem cells reflects their expression of receptors for chemokines and growth factors

The homing properties of adipose tissue-derived mesenchymal stem cells (AdMSCs) have stimulated intravenous applications for their use in stem cell therapy. However, the soluble factors and corresponding cellular receptors responsible for inducing chemotaxis of AdMSCs have not yet been reported. In the present study, the migration capacity of human AdMSCs (hAdMSCs) toward various cytokines or growth factors (GFs) and the expression of their receptors were determined. In a conventional migration assay, PDGF-AB, TGF-β1, and TNF-α showed the most effective chemoattractant activity. When AdMSCs were preincubated with various chemokines or GF, and then allowed to migrate toward medium containing 10% FBS, those preincubated with TNF-α showed the highest migratory activity. Next, hAdMSCs were either preincubated or not with TNF-α, and allowed to migrate in response to various GFs or chemokines. Prestimulation with TNF-α increased the migration activity of hAdMSCs compared to unstimulated hAdMSCs. When analyzed by FACS and RT-PCR methods, hAdMSCs were found to express C-C chemokine receptor type 1 (CCR1), CCR7, C-X-C chemokine receptor type 4 (CXCR4), CXCR5, CXCR6, EGF receptor, fibroblast growth factor receptor 1, TGF-β receptor 2, TNF receptor superfamily member 1A, PDGF receptor A and PDGF receptor B at both the protein and the mRNA levels. These results indicate that the migration capacity of hAdMSCs is controlled by various GFs and chemokines. Prior in vitro modulation of the homing capacity of hAdMSCs could stimulate their movement into injured sites in vivo when administered intravenously, thereby improving their therapeutic potential.     

Simvastatin inhibits sphingosylphosphorylcholine-induced differentiation of human mesenchymal stem cells into smooth muscle cells

Sphingosylphosphorylcholine (SPC) induces differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) into smooth muscle-like cells expressing α-smooth muscle actin (α-SMA) via transforming growth factor-β1/Smad2- and RhoA/Rho kinase-dependent mechanisms. 3-Hydroxy-3-methylglutaryl- coenzyme A reductase inhibitors (statins) have been known to have beneficial effects in the treatment of cardiovascular diseases. In the present study, we examined the effects of simvastatin on the SPC-induced α-SMA expression and Smad2 phosphorylation in hASCs. Simvastatin inhibited the SPC-induced α-SMA expression and sustained phosphorylation of Smad2 in hASCs. SPC treatment caused RhoA activation via a simvastatin-sensitive mechanism. The SPC-induced α-SMA expression and Smad2 phosphorylation were abrogated by pretreatment of the cells with the Rho kinase inhibitor Y27632 or overexpression of a dominant negative RhoA mutant. Furthermore, SPC induced secretion of TGF-β1 and pretreatment with either Y27632 or simvastatin inhibited the SPC-induced TGF-β1 secretion. These results suggest that simvastatin inhibits SPC-induced differentiation of hASCs into smooth muscle cells by attenuating the RhoA/Rho kinase-dependent activation of autocrine TGF-β1/Smad2 signaling pathway.