Smart Thermoresponsive Surfaces Based on pNIPAm Coatings and Laser Method for Biological Applications

Various applications within last decades such as bacterially resistant surfaces, soft robotics, drug delivery systems, sensors and tissue engineering are poised to feature the importance of the ability to control bio-interfacial interactions. An enhanced attention is dedicated to designing smart stimuli-responsive interfaces for DNA, drug delivery, protein and cell based applications. Within this context, the thermoresponsive materials, especially poly(N-isopropylacrylamide) (pNIPAm) have been intensively used in tissue engineering applications for a controlled detachment of proteins and cells with a minimum of invasive effect on protein and cell structural conformation. The properties of smart bio-interfaces can be controlled by its composition and polymer architecture. Therefore, appropriate methods for obtaining controlled coatings are necessary. Laser methods were successfully used in the last decades for obtaining controlled organic and inorganic coatings for various types of applications, from electronics to tissue engineering. Among these, Matrix-Assisted Pulsed Laser Evaporation (MAPLE) technique bring us a step forward to other laser methods by avoiding damage and photochemical decomposition of materials. In this chapter we describe materials and approaches used for design of smart bio-interfaces aimed at controlling protein and cells behavior in vitro, focusing MAPLE method for tuning coatings characteristics in relation with biological response

K2 Transfection System Boosts the Adenoviral Transduction of Murine Mesenchymal Stromal Cells

Adenoviral vectors are important vehicles for delivering therapeutic genes into mammalian cells. However, the yield of the adenoviral transduction of murine mesenchymal stromal cells (MSC) is low. Here, we aimed to improve the adenoviral transduction efficiency of bone marrow-derived MSC. Our data showed that among all the potential transduction boosters that we tested, the K2 Transfection System (K2TS) greatly increased the transduction efficiency. After optimization of both K2TS components, the yield of the adenoviral transduction increased from 18% to 96% for non-obese diabetic (NOD)-derived MSC, from 30% to 86% for C57BL/6-derived MSC, and from 0.6% to 63% for BALB/c-derived MSC, when 250 transduction units/cell were used. We found that MSC derived from these mouse strains expressed different levels of the coxsackievirus and adenovirus receptors (MSC from C57BL/6≥NOD>>>BALB/c). K2TS did not increase the level of the receptor expression, but desensitized the cells to foreign DNA and facilitated the virus entry into the cell. The expression of Stem cells antigen-1 (Sca-1) and 5′-nucleotidase (CD73) MSC markers, the adipogenic and osteogenic differentiation potential, and the immunosuppressive capacity were preserved after the adenoviral transduction of MSC in the presence of the K2TS. In conclusion, K2TS significantly enhanced the adenoviral transduction of MSC, without interfering with their main characteristics and properties

K2 Transfection System Boosts the Adenoviral Transduction of Murine Mesenchymal Stromal Cells

Adenoviral vectors are important vehicles for delivering therapeutic genes into mammalian cells. However, the yield of the adenoviral transduction of murine mesenchymal stromal cells (MSC) is low. Here, we aimed to improve the adenoviral transduction efficiency of bone marrow-derived MSC. Our data showed that among all the potential transduction boosters that we tested, the K2 Transfection System (K2TS) greatly increased the transduction efficiency. After optimization of both K2TS components, the yield of the adenoviral transduction increased from 18% to 96% for non-obese diabetic (NOD)-derived MSC, from 30% to 86% for C57BL/6-derived MSC, and from 0.6% to 63% for BALB/c-derived MSC, when 250 transduction units/cell were used. We found that MSC derived from these mouse strains expressed different levels of the coxsackievirus and adenovirus receptors (MSC from C57BL/6≥NOD>>>BALB/c). K2TS did not increase the level of the receptor expression, but desensitized the cells to foreign DNA and facilitated the virus entry into the cell. The expression of Stem cells antigen-1 (Sca-1) and 5′-nucleotidase (CD73) MSC markers, the adipogenic and osteogenic differentiation potential, and the immunosuppressive capacity were preserved after the adenoviral transduction of MSC in the presence of the K2TS. In conclusion, K2TS significantly enhanced the adenoviral transduction of MSC, without interfering with their main characteristics and properties

Treatment with Mesenchymal Stromal Cells Overexpressing Fas-Ligand Ameliorates Acute Graft-versus-Host Disease in Mice

Allogeneic hematopoietic cell transplantation (allo-HCT) has the potential to cure malignant and non-malignant hematological disorders, but because of the serious side effects of this intervention its applications are limited to a restricted number of diseases. Graft-versus-host disease (GvHD) is the most frequent complication and the leading cause of mortality and morbidity following allo-HCT. It results from the attack of the transplanted T cells from the graft against the cells of the recipient. There is no clear treatment for this severe complication. Due to their immunomodulatory properties, mesenchymal stromal cells (MSC) have been proposed to treat GvHD, but the results did not meet expectations. We have previously showed that the immunomodulatory effect of the MSC was significantly enhanced through adenoviral-mediated overexpression of FasL. In this study, we have tested the properties of FasL-overexpressing MSC in vivo, in a mouse model for acute GvHD. We found that treatment with FasL-overexpressing MSC delayed the onset of the disease and increased survival of the mice

Enhanced Suppression of Immune Cells In Vitro by MSC Overexpressing FasL

Mesenchymal stromal cells (MSC) display several mechanisms of action that may be harnessed for therapeutic purposes. One of their most attractive features is their immunomodulatory activity that has been extensively characterized both in vitro and in vivo. While this activity has proven to be very efficient, it is transient. We aimed to enhance it by transforming MSC to overexpress a first apoptosis signal (Fas) ligand (FasL). In this study, our goal was to induce FasL overexpression through adenoviral transduction in MSC to improve their immunomodulatory activity. We characterized the impact of FasL overexpression on the morphology, proliferation, viability, phenotype, multilineage differentiation potential and immunomodulation of MSC. Moreover, we determined their suppressive properties in mixed reactions with A20 cells, as well as with stimulated splenocytes. Our findings demonstrate that FasL-overexpressing MSC exhibit improved immunosuppressive properties, while maintaining their MSC-characteristic features. In conclusion, we establish, in a proof-of-concept set-up, that FasL-overexpressing MSC represent good candidates for therapeutic intervention targeted at autoimmune disorders

New Poly(N-isopropylacrylamide-butylacrylate) Copolymer Biointerfaces and Their Characteristic Influence on Cell Behavior In Vitro

Designing and obtaining new synthetic smart biointerfaces with specific and controlled characteristics relevant for applications in biomedical and bioengineering domains represents one of the main challenges in these fields. In this work, Matrix-Assisted Pulsed Laser Evaporation (MAPLE) is used to obtain synthetic biointerfaces of poly(N-isopropyl acrylamide-butyl acrylate) p(NIPAM-BA) copolymer with different characteristics (i.e., roughness, porosity, wettability), and their effect on normal HEK 293 T and murine melanoma B16-F1 cells is studied. For this, the influence of various solvents (chloroform, dimethylsulfoxide, water) and fluence variation (250–450 mJ/cm2) on the morphological, roughness, wettability, and physico–chemical characteristics of the coatings are evaluated by atomic force microscopy, scanning electron microscopy, contact angle measurements, Fourier-transform-IR spectroscopy, and X-ray photoelectron spectroscopy. Coatings obtained by the spin coating method are used for reference. No significant alteration in the chemistry of the surfaces is observed for the coatings obtained by both methods. All p(NIPAM-BA) coatings show hydrophilic character, with the exception of those obtained with chloroform at 250 mJ/cm2. The surface morphology is shown to depend on both solvent type and laser fluence and it ranges from smooth surfaces to rough and porous ones. Physico–chemical and biological analysis reveal that the MAPLE deposition method with fluences of 350–450 mJ/cm2 when using DMSO solvent is more appropriate for bioengineering applications due to the surface characteristics (i.e., pore presence) and to the good compatibility with normal cells and cytotoxicity against melanoma cells

Apolipoprotein A-II, a Player in Multiple Processes and Diseases

Apolipoprotein A-II (apoA-II) is the second most abundant apolipoprotein in high-density lipoprotein (HDL) particles, playing an important role in lipid metabolism. Human and murine apoA-II proteins have dissimilar properties, partially because human apoA-II is dimeric whereas the murine homolog is a monomer, suggesting that the role of apoA-II may be quite different in humans and mice. As a component of HDL, apoA-II influences lipid metabolism, being directly or indirectly involved in vascular diseases. Clinical and epidemiological studies resulted in conflicting findings regarding the proatherogenic or atheroprotective role of apoA-II. Human apoA-II deficiency has little influence on lipoprotein levels with no obvious clinical consequences, while murine apoA-II deficiency causes HDL deficit in mice. In humans, an increased plasma apoA-II concentration causes hypertriglyceridemia and lowers HDL levels. This dyslipidemia leads to glucose intolerance, and the ensuing high blood glucose enhances apoA-II transcription, generating a vicious circle that may cause type 2 diabetes (T2D). ApoA-II is also used as a biomarker in various diseases, such as pancreatic cancer. Herein, we provide a review of the most recent findings regarding the roles of apoA-II and its functions in various physiological processes and disease states, such as cardiovascular disease, cancer, amyloidosis, hepatitis, insulin resistance, obesity, and T2D

The Mechanism of Bisphenol A Atherogenicity Involves Apolipoprotein A-I Downregulation through NF-κB Activation

Apolipoprotein A-I (apoA-I) is the major protein component of high-density lipoproteins (HDL), mediating many of its atheroprotective properties. Increasing data reveal the pro-atherogenic effects of bisphenol A (BPA), one of the most prevalent environmental chemicals. In this study, we investigated the mechanisms by which BPA exerts pro-atherogenic effects. For this, LDLR−/− mice were fed with a high-fat diet and treated with 50 µg BPA/kg body weight by gavage. After two months of treatment, the area of atherosclerotic lesions in the aorta, triglycerides and total cholesterol levels were significantly increased, while HDL-cholesterol was decreased in BPA-treated LDLR−/− mice as compared to control mice. Real-Time PCR data showed that BPA treatment decreased hepatic apoA-I expression. BPA downregulated the activity of the apoA-I promoter in a dose-dependent manner. This inhibitory effect was mediated by MEKK1/NF-κB signaling pathways. Transfection experiments using apoA-I promoter deletion mutants, chromatin immunoprecipitation, and protein-DNA interaction assays demonstrated that treatment of hepatocytes with BPA induced NF-κB signaling and thus the recruitment of p65/50 proteins to the multiple NF-κB binding sites located in the apoA-I promoter. In conclusion, BPA exerts pro-atherogenic effects downregulating apoA-I by MEKK1 signaling and NF-κB activation in hepatocytes

Adenovirus-Mediated FasL Minigene Transfer Endows Transduced Cells with Killer Potential

Fas ligand (First apoptosis signal ligand, FasL, also known as CD95L) is the common executioner of apoptosis within the tumor necrosis factor (TNF) superfamily. We aimed to induce functional FasL expression in transduced cells using an adenovirus vector, which has the advantage of strong and transient induction of the gene included in the adenoviral genome. Here, we report that the adenovirus carrying a truncated FasL gene, named FasL minigene, encoding the full-length FasL protein (Ad-gFasL) is more efficient than the adenovirus carrying FasL cDNA (Ad-cFasL) in the induction of FasL expression in transduced cells. FasL minigene (2887 bp) lacking the second intron and a part of the 3′-UTR was created to reduce the gene length due to the size limitation of the adenoviral genome. The results show that, in transduced hepatocytes, strong expression of mRNA FasL appeared after 10 h for Ad-gFasL, while for Ad-cFasL, a faint expression appeared after 16 h. For Ad-gFasL, the protein expression was noticed starting with 0.5 transfection units (TU)/cell, while for Ad-cFasL, it could not be revealed. FasL-expressing endothelial cells induced apoptosis of A20 cells in co-culture experiments. FasL-expressing cells may be exploitable in various autoimmune diseases such as graft-versus-host disease, chronic colitis, and type I diabetes