Endothelial and mural laminin-α5 contributes to neurovascular integrity maintenance.

BACKGROUND: Laminin-α5, a major component of the basal lamina, is predominantly synthesized by endothelial and mural cells (pericytes and vascular smooth muscle cells) in the CNS. Loss of laminin-α5 in either population fails to induce any abnormalities due to functional redundancy. Thus, the functional significance of laminin-α5 in neurovascular integrity remains unknown. Here, we hypothesize that ablation of laminin-α5 in both endothelial and mural cells increases neurovascular permeability. METHODS: The compound knockout mice were generated by crossing laminin-α5 floxed mice with Tie2-Cre and PDGFRß-Cre, which target endothelial cells and mural cells, respectively. Neurovascular permeability in these mutants was determined with both exogenous and endogenous tracers. Endothelial paracellular and transcellular permeability was assessed by examining the expression of tight junction proteins and transcytosis-associated proteins. In addition, transmission electron microscopy (TEM) was used to visualize tight junction ultrastructure and endothelial caveolae vesicles. Defects in pericytes and astrocytes were investigated by examining pericyte coverage/contact and astrocyte polarity. RESULTS: Elevated neurovascular permeability was observed in the mutants. Subsequent studies found increased Caveolin-1 and decreased major facilitator superfamily domain-containing protein 2a (MFSD2A) expression, but unaltered Claudin-5 or zonula occludens-1 (ZO-1) expression. Consistent with these results, mutant mice exhibited increased endothelial caveolae vesicle number with intact tight junction structure under TEM. Additionally, pericyte coverage and contact were also decreased in the mutant mice, while astrocyte polarity was unaffected. CONCLUSIONS: These results strongly indicate that endothelial and mural cell-derived laminin-α5 actively maintains neurovascular integrity via the transcellular rather than paracellular mechanism.

A dispensable role of oligodendrocyte-derived laminin-α5 in brain homeostasis and intracerebral hemorrhage.

Laminin, a major component of the basal lamina in the CNS, is also expressed in oligodendrocytes (OLs). However, the function of OL-derived laminin remains largely unknown. Here, we performed loss-of-function studies using two OL-specific laminin-α5 conditional knockout mouse lines. Both mutants were grossly normal and displayed intact blood-brain barrier (BBB) integrity. In a mouse model of intracerebral hemorrhage (ICH), control mice and both mutants exhibited comparable hematoma size and neurological dysfunction. In addition, similar levels of hemoglobin and IgG leakage were detected in the mutant brains compared to the controls, indicating comparable BBB damage. Consistent with this finding, subsequent studies revealed no differences in tight junction protein (TJP) and caveolin-1 expression among control and knockout mice, suggesting that neither paracellular nor transcellular mechanism was affected in the mutants. Furthermore, compared to the controls, both mutant lines showed comparable oligodendrocyte number, oligodendrocyte proliferation rate, MBP/MAG levels, and SMI-32 expression, highlighting a minimal role of OL-derived laminin-α5 in OL biology. Together, these findings highlight a dispensable role of OL-derived laminin-α5 in both brain homeostasis and ICH pathogenesis.

Magnetic Hydroxyapatite Composite Nanoparticles for Augmented Differentiation of MC3T3-E1 Cells for Bone Tissue Engineering.

Progressive aging harms bone tissue structure and function and, thus, requires effective therapies focusing on permanent tissue regeneration rather than partial cure, beginning with regenerative medicine. Due to advances in tissue engineering, stimulating osteogenesis with biomimetic nanoparticles to create a regenerative niche has gained attention for its efficacy and cost-effectiveness. In particular, hydroxyapatite (HAP, Ca10(PO4)6(OH)2) has gained significant interest in orthopedic applications as a major inorganic mineral of native bone. Recently, magnetic nanoparticles (MNPs) have also been noted for their multifunctional potential for hyperthermia, MRI contrast agents, drug delivery, and mechanosensitive receptor manipulation to induce cell differentiation, etc. Thus, the present study synthesizes HAP-decorated MNPs (MHAP NPs) via the wet chemical co-precipitation method. Synthesized MHAP NPs were evaluated against the preosteoblast MC3T3-E1 cells towards concentration-dependent cytotoxicity, proliferation, morphology staining, ROS generation, and osteogenic differentiation. The result evidenced that MHAP NPs concentration up to 10 µg/mL was non-toxic even with the time-dependent proliferation studies. As nanoparticle concentration increased, FACS apoptosis assay and ROS data showed a significant rise in apoptosis and ROS generation. The MC3T3-E1 cells cocultured with 5 µg/mL MHAP NPs showed significant osteogenic differentiation potential. Thus, MHAP NPs synthesized with simple wet chemistry could be employed in bone regenerative therapy.

Highly efficient visible light driven photocatalytic activity of zinc/ferrite: Carbamazepine degradation, mechanism and toxicity assessment.

In this present study, spherical shaped zinc ferrite (Zn/Fe2O4) was prepared as uniformly sized (65 ± 0.5 nm) nanoparticles with band gap (2.00 eV) in a visible light regime and employed for the photocatalytic degradation of carbamazepine (CBZ). The doping of Zn decreased the band gap (from 2.00 to 1.98 eV) and enhanced the absorption of visible light. Zinc doping also induced effective separation of photogenerated carriers and subsequent charge migration to the surface of the Zn/Fe2O4 nanoparticle. On account of the advantages of the material, a high removal efficiency (~ 100%) of CBZ through photocatalytic degradation was achieved. Kinetics of CBZ degradation follows a pseudo first-order with the rate constant 0.0367 min-1. In-vitro and in-vivo toxicity of the nanoparticles were examined promoting the environmental implications.

IGF-1 Facilitates Cartilage Reconstruction by Regulating PI3K/AKT, MAPK, and NF-kB Signaling in Rabbit Osteoarthritis.

PURPOSE: The pathogenesis of osteoarthritis (OA) is characterized by joint degeneration. The pro-inflammatory cytokine interleukin (IL)-1ß plays a vital role in the pathogenesis of OA by stimulation of specific signaling pathways like NF-κB, PI3K/Akt, and MAPKs pathways. The catabolic role of growth factors in the OA may be inhibited cytokine-activated pathogen. The purpose of this study was to investigate the potential effects of insulin-like growth factor-1 (IGF-1) on IL-1ß-induced apoptosis in rabbit chondrocytes in vitro and in an in vivo rabbit knee OA model. METHODS: In the present study, the OA developed in chondrocyte with the treatment of IL-1ß and articular cartilage ruptures by removal of cartilage from the rabbit knee femoral condyle. After IGF-1 treatment, immunohistochemistry and qRT-PCR were identified OA expression with changes in MMPs (matrix metalloproteinases). The production of ROS (intracellular reactive oxygen species) in the OA was detected by flow cytometry. Further, the disease progression was microscopically investigated and pathophysiological changes were analyzed using histology. The NF-κB, PI3K/Akt and P38 (MAPK) specific pathways that are associated with disease progression were also checked using the Western blot technique. RESULTS: The expression of MMPs and various apoptotic markers are down-regulated following administration of IGF-1 in a dose-dependent fashion while significantly up-regulation of TIMP-1. The results showed that higher levels of ROS were observed upon treatment of chondrocytes and chondral OA with IL-1ß. Collectively, our results indicated that IGF-1 protected NF-κB pathway by suppression of PI3K/Akt and MAPKs specific pathways. Furthermore, the macroscopic and pathological investigation showed that it has a chondroprotective effect by the formation of hyaline cartilage. CONCLUSION: Our results indicate a protective effect of IGF-1 against OA pathogenesis by inhibition of NF-κB signaling via regulation of the MAPK and PI3K/Akt signaling pathways and prevention of apoptosis by suppression of ROS production.

A gastric cancer cell derived extracellular compounds suppresses CD161+CD3- lymphocytes and aggravates tumor formation in a syngeneic mouse model.

Evasion of the immune system is often associated with malignant tumors. The cancer cell microenvironment plays an important role in tumor progression, but its mechanism is largely unknown. Here we show that an extracellular compound derived from gastric cancer (GC-EC) selectively suppresses CD161+CD3- natural killer (NK) cells. Splenocytes treated with GC-EC showed considerable proliferation and the CD161+CD3- NK cell population was time-dependently suppressed. Intracellular staining of IFN-γ was shown to be down-regulated in concert with granzyme B and perforin. A cytotoxicity assay of splenocytes treated with GC-EC against K-562 cells showed a significant reduction in cytolytic activity. Further, the immune-suppressive effect of GC-EC was more evident in a syngeneic tumor model in C57BL/6 mice. Animals treated with B16 F10 and GC-EC exhibited more aggravated tumor formation than animals treated with B16 F10 only. We demonstrated that inhibition of apoptosis while increasing PI3 K/AKT levels may provoke tumor formation by GC-EC. A cytokine array revealed the presence of several cytokines in GC-EC that negatively regulate immune cytolytic activity and could be potential candidates for immune-suppressive effects.

Ginseng compound K incorporated porous Chitosan/biphasic calcium phosphate composite microsphere for bone regeneration.

There is a substantial for the bone graft materials in the clinical field. Porous, stable and biodegradable bone microsphere scaffold using biopolymer chitosan was studied, and biphasic calcium phosphate was added to improve mechanical and osteoconductivity properties later ginseng compound K was added for improving its medicinal properties. They were characterized using FTIR and XRD that showed the apatite crystal in the composite microsphere scaffolds were structurally similar to that of biogenic apatite crystals. Scanning electron microscopy images confirmed the presence of hydroxyapatite on the surface of the composite microspheres. In vitro results infers that the composite microspheres are biocompatible with NIH 3T3 and MG63 cells and capable of supporting growth and spreading of MG-63 cells. Further, Osteogenic markers expression was found to be higher in rat bone marrow stem cells seeded on microsphere scaffolds compared to control. The prepared biocomposite porous microsphere scaffold developed in this study can be used as an alternative for the bone regeneration or bone tissue engineering.

Biological effects of cyclosporin A on CD3-CD161+ and CD3+CD161+ lymphocytes.

Cyclosporin A (CSA) is a widely used drug to prevent the immune cell function. It is well known that CSA blocks transcription of cytokine genes in activated T cells. The connection between T cells and CSA has been well established. However, the effect of CSA on natural killer (NK) cells is not thoroughly understood. Therefore, in the present study, splenocytes and peripheral blood mononuclear cells (PBMCs) were treated with CSA in the presence of concanavalin A (Con A) or interleukin-2 (IL-2). CSA at higher concentrations induces apoptosis and inhibition of proliferation, while lower concentrations showed synergistically enhanced proliferation in splenocytes and PBMCs. Further, CSA favored the in vitro conversion of CD3+CD161+ cells. Splenocytes and PBMC were found to have synergistic proliferation with Con A, and PBMC exhibited significantly higher expression of NKp30, NKp44, and granzyme B along with enhanced cytotoxicity against K-562 cells in CSA-treated animals. Proliferation assay also showed that proliferation of CD161+ cells was higher in CSA-treated animals. Collectively, our results suggest that CSA differentially influences the population, function, and expression of the NK cell phenotype.

Morphological Modification of Carbon Nanoparticles after Interacting with Methotrexate as a Potential Anticancer Agent.

PURPOSE: Production of highly penetrable and targetable drug delivery particles is mainly focused by current therapy and such focus is achieved in our present study. The carbon nanoparticle (CNP) prepared from purely natural source was modified from spherical shape to cylindrical floral like structure after treatment with the anticancer drug methotrexate (CM). METHODS: The physiochemical properties of the CNP and CM was characterized using FT-IR/Raman Spectrometer, XRD, SEM, AFM, particle size analyzer and its biological evaluation using haemolysis and MTT assay. RESULTS: The shift in FT-IR peaks at 1592, 1120 cm-1 and peaks of raman spectra observed at 1303, 1300 cm-1 represents ordered carbon nanotubes. The morphological change from spherical to cylindrical floral like structure was observed using SEM and AFM and its particle size distribution analysis shows an average diameter of 269 nm for CM. XRD peak at 2θ = 23.86° (002) indicates the presence of large amount of amorphous material that corresponds to multi-walled carbon nanotubes. Haemocompatibility studies proved the safety level usage as 100 µg/ml and MTT assay shows viability rate of 85-98% with mouse embryonic fibroblast (NIH/3 T3) and 30-45% with pancreatic carcinoma (MIA PaCa-2) and gastric cancer cell lines (SNU- 484) respectively.These results are also supported by phase contrast microscope images observed after staining with calcein AM and EthD-1. CONCLUSIONS: The morphologically modified CNPs has shown good anticancer, biocompatibility and haemocompatibility property which is an important criterion to be satisfied by a biomedical product.

Aronia melanocarpa (Black Chokeberry) Reduces Ethanol-Induced Gastric Damage via Regulation of HSP-70, NF-κB, and MCP-1 Signaling.

Aronia melanocarpa (Michx.) Ell. belongs to the Rosaceae family. The purpose of this study is to explore the gastroprotective effect of the Aronia melanocarpa hydro-alcoholic extract (AMHAE) against ethanol-induced gastric ulcer in a rat model. Different concentrations (50, 100, and 200 mg/kg) of AMHAE, or 30 mg/kg of omeprazole, significantly inhibited the gastric injury formation. The ethanol-induced ulcer group showed significant increases of malondialdehyde (MDA), myeloperoxidase (MPO), tumor necrosis factor (TNF)-α, nuclear factor-kappaB p65 (NF-κB p65), and monocyte chemoattractant protein (MCP)-1, and decreased activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-px), and interleukin (IL)-4. However, AMHAE (200 mg/kg) pretreatment significantly reversed the altered pathophysiological levels of these biomolecules to near normal stages. The gastroprotective activity of AMHAE was abolished by pretreatment with l-NAME, naloxone, capsazepine, and indomethacin, demonstrating the participation of nitric oxide (NO), opioids, TRPV (vanilloid receptor-related transient receptor potential), and prostaglandins in AMHAE-assisted gastroprotection against ethanol-induced gastric injuries. This gastroprotective effect of AMHAE might be due to the downregulation of TNF-α-based NF-κB, MCP-1 signaling and strong antioxidant properties.

Reengineered graft copolymers as a potential alternative for the bone tissue engineering application by inducing osteogenic markers expression and biocompatibility.

Composite scaffolds of nano-hydroxyapatite with demineralized bone matrix were prepared and they were graft copolymerized for better bone regeneration and drug delivery applications. The graft copolymers were characterized for their physiochemical properties using conventional methods like FTIR, TGA, XRD and SEM. The scaffolds were seeded with 3T3 and MG63 cells for studying their biocompatibility and their temporal expression of ALP activity, the rate of calcium deposition and their gene expression of collagen type I (Coll-1), osteopontin (OP), osteonectin (ON), and osteocalcin (OC) were studied. In vivo studies were conducted using sub-cutaneous implantation models in male Wister rats for 6 months. Periodic radiography and post-autopsy histopathology was analysed at 15days, 1, 2, 3, 4, 5, and 6 months. The obtained in vitro results clearly confirm that the bone scaffolds prepared in this study are biocompatible, superior osteoinductivity, capable of supporting growth, maturation of MG 63 osteoblast like cells; the gene expression profile revealed that the material is capable of supporting the in vitro growth and maturation of osteoblast-like cells and maturation. The in vivo results stand a testimony to the in vitro results in proving the biocompatibility and osteoinductivity of the materials.