Anti-Fibronectin 1 (F2372) Antibody (A25667)

Islet transplantation is considered to be a curative treatment for type 1 diabetes mellitus. However, disruption of the extracellular matrix (ECM) leads to β-cell destruction and graft dysfunction. In this study, we developed a functionalized self-assembling peptide, KLD-F, with ECM mimic motifs derived from fibronectin and collagen IV, and evaluated its effect on β-cell function and proliferation. Atomic force microscopy and rheological results showed that KLD-F could self-assemble into a nanofibrous scaffold and change into a hydrogel in physiological saline condition. In a three-dimensional cell culture model, KLD-F improved ECM remodeling and cell-cell adhesion of INS-1 β-cells by upregulation of E-cadherin, fibronectin, and collagen IV. KLD-F also enhanced glucose-stimulated insulin secretion and expression of β-cell function genes, including Glut2, Ins1, MafA, and Pdx-1 in INS-1 cells. Moreover, KLD-F promoted proliferation of INS-1 β-cells and upregulated Ki67 expression by mediating cell cycle progression. In addition, KLD-F improved β-cell function and proliferation via an integrin/focal adhesion kinase/extracellular signal-regulated kinase/cyclin D pathway. This study highlights the fact that the β-cell-ECM interaction reestablished with this functionalized self-assembling peptide is a promising method to improve the therapeutic efficacy of islet transplantation.

The present study aimed to investigate whether klotho gene delivery attenuated renal hypertrophy and fibrosis in streptozotocin-induced diabetic rats. A recombinant adeno-associated virus (rAAV) carrying mouse klotho full-length cDNA (rAAV.mKL), was constructed for in vivo investigation of klotho expression. Diabetes was induced in rats by a single tail vein injection of 60 mg/kg streptozotocin. Subsequently, the diabetic rats received an intravenous injection of rAAV.mKL, rAAV.green fluorescent protein (GFP) or phosphate-buffered saline (PBS). The Sprague-Dawley rat group received PBS and served as the control group. After 12 weeks, all the rats were sacrificed and ELISA, immunohistochemical and histological analyses, fluorescence microscopy, semi-quantitative reverse transcription-polymerase chain reaction and western blottin were performed. A single dose of rAAV.mKL was found to prevent the progression of renal hypertrophy and fibrosis for at least 12 weeks (duration of study). Klotho expression was suppressed in the diabetic rats, but was increased by rAAV.mKL delivery. rAAV.mKL significantly suppressed diabetes-induced renal hypertrophy and histopathological changes, reduced renal collagen fiber generation and decreased kidney hypertrophy index. In addition, rAAV.mKL decreased the protein expression levels of fibronectin and vimentin, while it downregulated the mRNA expression and activity of Rho-associated coiled-coil kinase (ROCK)I in the kidneys of the diabetic rats. These results indicated that klotho gene delivery ameliorated renal hypertrophy and fibrosis in diabetic rats, possibly by suppressing the ROCK signaling pathway. This may offer a novel approach for the long-term control and renoprotection of diabetes.

Epithelial-mesenchymal transition (EMT) plays a specific role in the migration of tumor cells. Both estrogen and midkine (MK) have been thought to be important factors in promoting the progression of non-small-cell lung cancer (NSCLC) and can enhance EMT. Some evidence indicated the correlation between estradiol (E2) and MK, but the precise mechanism on their interreaction is unknown. Here, we try to clarify whether and how E2 regulates MK expression to promote EMT. We found that E2 increased MK mRNA expression in lung adenocarcinoma cells LTEP-a2 and A549 in a time-dependent manner. E2-induced MK expression was inhibited by the estrogen receptor (ER) antagonist ICI 182,780 and tamoxifen but not by phosphoinositide-3 kinase and MAPK inhibitors, suggesting a genomic mechanism of E2 on the regulation of MK transcription. Moreover, luciferase reporter and chromatin immunoprecipitation assays exhibited that E2 induced ERβ recruitment to the estrogen response element in the MK promoter. Small interfering RNA to ERα and ERβ revealed that ERβ mainly mediated E2-induced MK transcription. Interestingly, E2 enhanced MK expression in accordance with increase of EMT, whereas knockdown of MK could block EMT under E2 stimulation. Importantly, through analyzing lung adenocarcinoma tissues, there was indeed a correlation among levels of E2, MK, and EMT-related protein expression. Taken together, we reported a previously unrecognized mechanism on E2 in the regulation of MK expression and proved that MK plays a pivotal role in progression of E2-regulated EMT.

Cancer stem cells (CSCs) are a subpopulation of neoplastic cells with self-renewal capacity and limitless proliferative potential as well as high invasion and migration capacity. These cells are commonly associated with epithelial-mesenchymal transition (EMT), which is also critical for tumor metastasis. Recent studies illustrate a direct link between EMT and stemness of cancer cells. Long non-coding RNAs (lncRNAs) have emerged as important new players in the regulation of multiple cellular processes in various diseases. To date, the role of lncRNAs in EMT-associated CSC stemness acquisition and maintenance remains unclear. In this study, we discovered that a set of lncRNAs were dysregulated in Twist-positive mammosphere cells using lncRNA microarray analysis. Multiple lncRNAs-associated canonical signaling pathways were identified via bioinformatics analysis. Especially, the Shh-GLI1 pathway associated lncRNA-Hh, transcriptionally regulated by Twist, directly targets GAS1 to stimulate the activation of hedgehog signaling (Hh). The activated Hh increases GLI1 expression, and enhances the expression of SOX2 and OCT4 to play a regulatory role in CSC maintenance. Thus, the mammosphere-formation efficiency (MFE) and the self-renewal capacity in vitro, and oncogenicity in vivo in Twist-positive breast cancer cells are elevated. lncRNA-Hh silence in Twist-positive breast cells attenuates the activated Shh-GLI1 signaling and decreases the CSC-associated SOX and OCT4 levels, thus reduces the MFE and tumorigenesis of transplanted tumor. Our results reveal that lncRNAs function as an important regulator endowing Twist-induced EMT cells to gain the CSC-like stemness properties.

Lumbar spinal stenosis (LSS) is a syndromic degenerative spinal disease and is characterized by spinal canal narrowing with subsequent neural compression causing gait disturbances. Although LSS is a major age-related musculoskeletal disease that causes large decreases in the daily living activities of the elderly, its molecular pathology has not been investigated using proteomics. Thus, we used several proteomic technologies to analyze the ligamentum flavum (LF) of individuals with LSS. Using comprehensive proteomics with strong cation exchange fractionation, we detected 1288 proteins in these LF samples. A GO analysis of the comprehensive proteome revealed that more than 30% of the identified proteins were extracellular. Next, we used 2D image converted analysis of LC/MS to compare LF obtained from individuals with LSS to that obtained from individuals with disc herniation (nondegenerative control). We detected 64 781 MS peaks and identified 1675 differentially expressed peptides derived from 286 proteins. We verified four differentially expressed proteins (fibronectin, serine protease HTRA1, tenascin, and asporin) by quantitative proteomics using SRM/MRM. The present proteomic study is the first to identify proteins from degenerated and hypertrophied LF in LSS, which will help in studying LSS.

More and more evidences indicate that endocrine disruptor chemicals such as bisphenol A (BPA) can act as carcinogens and enhance susceptibility to tumorigenesis. Although the gut is in direct contact with orally ingested BPA, effects of BPA on occurrence and development of colorectal cancer remain an unexplored endpoint. Colorectal cancer SW480 cells treated with nanomolar (10(-8) M) or greater (10(-5) M) concentrations of BPA were compared with responses of a control group. Proteomic study revealed that more than 56 proteins were modulated following exposure to BPA, which are relevant to structure, motility and proliferation of cells, production of ATP, oxidative stress, and protein metabolism. Further studies revealed that BPA increased migration and invasion and triggered transformations from epithelial to mesenchymal transitions (EMTs) of colorectal cancer cells, which was characterized by acquiring mesenchymal spindle-like morphology and increasing the expression of N-cadherin with a concomitant decrease of E-cadherin. Accordingly, BPA treatment increased the expression of transcription factor Snail. Furthermore, signal AKT/GSK-3β-mediated stabilization of Snail is involved during BPA-induced EMT of colon cancer cells. Our study first demonstrated that the xenoestrogen BPA at nanomolar and greater concentrations modulates the protein profiles and promotes the metastasis of colorectal cancer cells via induction of EMT.

The epithelial-to-mesenchymal transient (EMT) is associated with tumor metastasis. Twist is one of the key transcription factors for EMT and relates to tumor cell migration. Long non-coding RNAs (lncRNAs) have recently emerged as important regulatory molecules involved in a broad range of biological processes and complicated diseases. However, it is unknown whether a signal network and lncRNAs are involved in Twist-induced EMT program. Taking MCF10A/Twist as a model, more than 99 lncRNAs and 3164 genes are regulated in the Twist-induced EMT process using lncRNA-array and cDNA micro-array. We establish a downstream signal network associated with EMT induced by Twist using bioinformatic analysis (Gene Ontology, pathway analysis) and experimental data. A set of multiple canonical signal pathways (such as WNT, MAPK, JAK/STAT, TGF-β, mTOR, Hedgehog and P53 signaling pathways) and several lncRNAs [such as lncRNA (chr6, 26124411-26139312, +), lncRNA (chr1, 41944445-41949874, -), lncRNA (chr17, 44833874-44834830, +)] are altered in MCF10A/Twist cells. More interestingly, lncRNA (chr17, 44833874-44834830, +), lncRNA (chr17, 21142183-21156578, -), lncRNA (chr6, 26124411-26139312, +) and lncRNA (chr19, 438420-2083745, -) may be involved in regulation or activation of WNT signaling pathway in the Twist-induced EMT process. These findings first determine that Twist contributes to invasion and metastasis by inducing wide-ranging transcriptional and functional changes of lncRNAs and signal pathways in our study.

This work developed a novel bi-layer wound dressing composed of 3D activated carbon fibers that allows facilitates fibroblast cell growth and migration to a wound site for tissue reconstruction, and the gentamicin is incorporated into a poly(γ-glutamic acid)/gelatin membrane to prevent bacterial infection. In an in vitro, field emission scanning electron microscopy shows that rat skin fibroblasts appeared and spread on the surface of activated carbon fibers, and penetrated the interior and exterior of the 3D activated carbon fiber construct to a depth of roughly 200 μm. An in vivo analysis shows that fibroblast cells containing the proposed 3D scaffold had the potential of a biologically functionalized dressing to accelerate wound closure. Additionally, fibroblasts migrated to the wound site in a bi-layer wound dressing containing fibroblasts, enhancing fibronectin and type I collagen expression, resulting in faster skin regeneration than that achieved with a Tegaderm™ hydrocolloid dressing or gauze.

BACKGROUND:

Neuroinflammation occurs in insulted regions of the brain and may be due to reactive oxygen species (ROS), nitric oxide (NO), cytokines, and chemokines produced by activated glia. Excessive production of neurotoxic molecules causes further neuronal damage. Low levels of vitamin D3 are a risk factor for various brain diseases.

METHODS:

Using the bacterial endotoxin, lipopolysaccharide (LPS), to induce neuroinflammation in primary cortical neuron-glia cultures, we investigated how 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) affected neuroinflammation.

RESULTS:

LPS (100 ng/ml) induced the accumulation of nitrite and the production of ROS, interleukin (IL)-6, and macrophage inflammatory protein (MIP)-2 in time-dependent manners. Inhibition of p38 and extracellular signal-regulated kinase (ERK) but not c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) by 20 μM of SB203580, PD98059, and SP600125, significantly reduced LPS-induced ROS production, NO accumulation, and inducible NO synthase (iNOS) expression, respectively. LPS-induced IL-6 and MIP-2 were significantly attenuated by inhibition of p38, ERK, and JNK MAPK. Cotreatment with 1,25(OH)2D3 attenuated LPS-induced ROS production, NO accumulation, and iNOS expression in concentration-dependent manners. 1,25(OH)2D3 also reduced LPS-induced production of IL-6 and MIP-2. Similarly, iNOS, IL-6, and MIP-2 mRNA expression in cells treated with LPS significantly increased, whereas this effect was attenuated by 1,25(OH)2D3. Moreover, LPS-induced phosphorylation of p38, ERK, and JNK MAPK was significantly inhibited by 1,25(OH)2D3.

CONCLUSIONS:

Our findings indicate that 1,25(OH)2D3 reduced the LPS-stimulated production of inflammatory molecules in neuron-glia cultures by inhibiting MAPK pathways and the production of downstream inflammatory molecules. We suggest that 1,25(OH)2D3 can be used to alleviate neuroinflammation in various brain injuries.