Preclinical evaluation of the safety and effectiveness of a new bioartificial cornea.

Cross-linking agents are frequently used to restore corneal properties after decellularization, and it is especially important to select an appropriate method to avoid excessive cross-linking. In addition, how to promote wound healing and how to improve scar formation require further investigation. To ensure the safety and efficacy of animal-derived products, we designed bioartificial corneas (BACs) according to the criteria for Class III medical devices. Our BACs do not require cross-linking agents and increase mechanical strength via self-cross-linking of aldehyde-modified hyaluronic acid (AHA) and carboxymethyl chitosan (CMC) on the surface of decellularized porcine corneas (DPCs). The results showed that the BACs had good biocompatibility and transparency, and the modification enhanced their antibacterial and anti-inflammatory properties in vitro. Preclinical animal studies showed that the BACs can rapidly regenerate the epithelium and restore vision within a month. After 3 months, the BACs were gradually filled with epithelial, stromal, and neuronal cells, and after 6 months, their transparency and histology were almost normal. In addition, side effects such as corneal neovascularization, conjunctival hyperemia, and ciliary body hyperemia rarely occur in vivo. Therefore, these BACs show promise for clinical application for the treatment of infectious corneal ulcers and as a temporary covering for corneal perforations to achieve the more time.

Construction of Antithrombotic Tissue-Engineered Blood Vessel via Reduced Graphene Oxide Based Dual-Enzyme Biomimetic Cascade.

Thrombosis is one of the biggest obstacles in the clinical application of small-diameter tissue-engineered blood vessels (TEBVs). The implantation of an unmodified TEBV will lead to platelet aggregation and further activation of the coagulation cascade, in which the high concentration of adenosine diphosphate (ADP) that is released by platelets plays an important role. Inspired by the phenomenon that endothelial cells continuously generate endogenous antiplatelet substances via enzymatic reactions, we designed a reduced graphene oxide (RGO) based dual-enzyme biomimetic cascade to successively convert ADP into adenosine monophosphate (AMP) and AMP into adenosine. We used RGO as a support and bound apyrase and 5'-nucleotidase (5'-NT) on the surface of RGO through covalent bonds, and then, we modified the surface of the collagen-coated decellularized vascular matrix with the RGO-enzyme complexes, in which RGO functions as a platform with a large open surface area and minimal diffusion barriers for substrates/products to integrate two catalytic systems for cascading reactions. The experimental results demonstrate that the two enzymes can synergistically catalyze procoagulant ADP into anticoagulant AMP and adenosine successively under physiological conditions, thus reducing the concentration of ADP. AMP and adenosine can weaken or even reverse the platelet aggregation induced by ADP, thereby inhibiting thrombosis. Adenosine can also accelerate the endothelialization of TEBVs by regulating cellular energy metabolism and optimizing the microenvironment, thus ensuring the antithrombotic function and patency of TEBVs even after the RGO-enzyme complex loses its activity.

Maintaining Moderate Platelet Aggregation and Improving Metabolism of Endothelial Progenitor Cells Increase the Patency Rate of Tissue-Engineered Blood Vessels.

Small-diameter tissue-engineered blood vessels (TEBVs) have been associated with low, long-term patency rates primarily because of acute thrombosis in early stages and an inability to achieve early endothelialization. Platelets and endothelial progenitor cells (EPCs) play a key role in these processes. A nano delayed-release 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR)-bound TEBV was implanted in rat carotid arteries for 3 months. AICAR-bound TEBVs had a high patency rate compared with control TEBVs after 3 months. We found that AICAR maintained moderate platelet aggregation in vivo. In vitro data indicated that AICAR inhibits the release of 5-hydroxytryptamine and thromboxane A2 in activating platelets to reduce platelet aggregation. Then, we confirmed that AICAR strengthens the EPC energy state, which results in earlier endothelialization. The homing, migration, and paracrine function of EPCs were enhanced by AICAR in vitro. Besides, AICAR can contribute to the migration of endothelial cells near the anastomosis. The cellularization of TEBVs at different time points was observed too. In conclusion, our study suggests that the application of nanodelivery material containing AICAR can effectively improve small-diameter TEBVs by maintaining moderate platelet aggregation and improving metabolism of EPCs.

Systematic profiling of mRNA and miRNA expression in the pancreatic islets of spontaneously diabetic Goto-Kakizaki rats.

Type 2 diabetes (T2DM) is a complex multifactorial metabolic disorder that affects >100 million individuals worldwide, yet the mechanisms involved in the development and progression of the disease have not yet been fully elucidated. The present study examined the mRNA and micro (mi)RNA expression profiles by microarray analysis in the pancreas islets of spontaneously diabetic Goto-Kakizaki rats with the aim to identify regulatory mechanisms underlying the pathogenesis of T2DM. A total of 9 upregulated and 10 downregulated miRNAs were identified, including miR-150, miR-497, miR-344-3p and let-7f, which were independently validated by quantitative polymerase chain reaction assays. In addition, differential expression of 670 genes was detected by mRNA microarray analysis, including 370 upregulated and 247 downregulated genes. The differentially expressed genes were statistically associated with major cellular pathways, including the immune response pathway and the extracellular matrix (ECM)-receptor interaction pathway. Finally, a reverse regulatory association of differentially expressed miRNAs and their predicted target genes was constructed, supported by analysis of their mRNA and miRNA expression profiles. A number of key pairs of miRNA-mRNA was proposed to have significant roles in the pathogenesis of T2DM rats based on bioinformatics analysis, one example being the let-7f/collagen, type II, alpha 1 pair that may regulate ECM-receptor interactions.

Systematic dielectrophoretic analysis of the Ara-C-induced NB4 cell apoptosis combined with gene expression profiling.

Dielectrophoresis (DEP) can be used to noninvasively measure the dielectric state of the cell, and this data can be used to monitor cell health or apoptosis. In this study, we followed events associated with cytosine arabinoside (Ara-C)-induced apoptosis in NB4 cells using DEP analysis. Our data showed that the membrane capacitance of NB4 cells decreases from 9.42 to 7.63 mF/m(2) in the first 2 hours following treatment with Ara-C, and that this decreased capacitance persists for >12 hours. Additionally, cytoplasmic conductivity decreases from 0.217 to 0.190 S/m within 2 hours of Ara-C treatment; this level is maintained for a short period of time before decreasing. We also investigated these events molecularly at the level of gene expression using microarray analysis and showed that the expression of genes related to membrane capacitance and cytoplasmic conductivity change dramatically as early as 2 hours post-Ara-C treatment, and further demonstrated a temporal relationship between the dielectric properties and key events in apoptosis. This study, integrating physical electrical properties of the cell membrane and cytoplasm with those of conductivity-related gene networks, provides new insights into the molecular mechanisms underlying the initiation of apoptosis, establishing a systematic foundation for DEP application in follow-up drug screening and development of medicines for treating leukemia.

Construction of a novel oligonucleotide array-based transcription factor interaction assay platform and its uses for profiling STAT1 cofactors in mouse fibroblast cells.

Here, we describe a novel oligonucleotide array-based transcription factor (TF) interaction assay platform that can directly identify cointeracting TF complexes following binding to their regulatory DNA elements. This platform that combines immuno-coprecipitation technology with our previously reported oligonucleotide array-based TF assay (OATFA), is named targeted immuno-coprecipitation OATFA (TIC-OATFA). We illustrate use of the system to identify interaction partners of STAT1 (signal transducer and activator of transcription proteins 1) in mouse fibroblasts. Several previously known partners of STAT1, as well as new partners, were identified by TIC-OATFA, including the upstream stimulatory factors 1 and 2 (USF1, USF2), nuclear factor of activated T cells, TATA box-binding protein, nuclear factor erythroid-derived 2, nuclear factor-kappa B, and nuclear factor 1. Both USF1 and nuclear factor-kappa B are well known to interact with STAT1, but the other five TFs are previously unreported STAT1 interaction partners. We examined interactions between one new TF, USF2, and STAT1 in detail. USF2 belongs to the group of bHLH-zip transcription factors, which in a number of diseases including cancers, has enhanced activity. In summary, a novel oligonucleotide array-based assay platform was developed and used to study interactions between STAT1 and functional TF binding partners, revealing that USF2 and potentially four other new TFs are partners of STAT1 in an IFN-γ stimulated mouse fibroblast cell line.

Broad profiling of DNA-binding transcription factor activities improves regulatory network construction in adult mouse tissues.

Molecular systematics involves the description of the regulatory networks formed by the interconnections between active transcription factors and their target expressed genes. Here, we have determined the activities of 200 different transcription factors in six mouse tissues using an advanced mouse oligonucleotide array-based transcription factor assay (MOUSE OATFA). The transcription factor signatures from MOUSE OATFA were combined with public mRNA expression profiles to construct experimental transcriptional regulatory networks in each tissue. SRF-centered regulatory networks constructed for lung and skeletal muscle with OATFA data were confirmed by ChIP assays, and revealed examples of novel networks of expressed genes coregulated by sets of transcription factors. The combination of MOUSE OATFA with bioinformatics analysis of expressed genes provides a new paradigm for the comprehensive prediction of the transcriptional systems and their regulatory pathways in mouse.