Poly-L-Lysine Solution (PLL), 1 mg/ml.

Background While the role of pericytes in blood-brain barrier (BBB) disruption and neuroinflammation is well-established in adult neurological disorders, their contribut... More

Background While the role of pericytes in blood-brain barrier (BBB) disruption and neuroinflammation is well-established in adult neurological disorders, their contribution to neonatal brain injury is largely unexplored. Here, we investigated the role of brain pericytes in hypoxic-ischemic (HI) brain injury in the developing brain, with a particular focus on the regulatory role of pericyte-derived microRNA210 (miR210) in pericyte dysfunction. Methods HI brain injury was induced on postnatal day 9 transgenic mice, including Atp13a5-tdTomato brain pericyte reporter mice, pericyte-specific diphtheria toxin receptor mice, miR210 knockout mice, and wild-type controls. Post-injury assessments include brain infarct, brain edema, BBB permeability, ELISA, western blotting, immunostaining, and neurological function test. BBB-associated cells, including pericytes and endothelial cells, were isolated from mouse brain using an immunomagnetic approach. RNA sequencing analysis was conducted to examine transcriptomic changes in pericytes after HI. To investigate the regulatory role of miR210 in pericyte dysfunction and its underlying mechanisms, primary pericytes were transfected with miR210 mimic or negative control, followed by oxygen-glucose deprivation. Transfected cells were also treated with either interleukin 1 type 1 receptor neutralizing antibody or recombinant interleukin 1 type 2 receptor chimera protein. Post-assays included RT-qPCR, immunostaining and cell viability assay. Student’s t test or one-way ANOVA followed by Bonferroni test was used, as appropriate. Results HI resulted in a time-dependent loss of pericytes in pericyte reporter mouse pups. Ablation of brain pericytes exacerbated BBB disruption and HI brain injury in neonatal brain. miR210 deletion mitigated brain pericyte loss and BBB leakage post-HI. Transcriptomic analysis revealed that HI-induced pericyte dysfunction was associated with upregulated genes enriched in biological processes such as “cellular response to interleukin 1”. miR210 knockout suppressed the expression of proinflammatory markers such as Il1r1. Mechanistically, miR210 overexpression increased proinflammatory cytokine levels and promoted pericyte cell death under oxygen-glucose deprivation, effects that were reversed by IL1R1 blockade. Importantly, brain pericyte-specific miR210 deletion preserved pericyte viability and BBB integrity, and provided neuroprotection after HI. Conclusions These findings underscore the critical role of brain pericytes in BBB function in the developing brain and identify miR210 as a central regulator of pericyte dysfunction following neonatal HI brain injury. Less

The serine protease tissue-type plasminogen activator (t-PA) is involved in both vital physiological brain processes, such as synaptic plasticity, and pathophysiological ... More

The serine protease tissue-type plasminogen activator (t-PA) is involved in both vital physiological brain processes, such as synaptic plasticity, and pathophysiological conditions, such as neurodegeneration and ischemic stroke. Recent data suggest that epigenetic mechanisms play an important role in the regulation of t-PA in human endothelial cells. However, there are limited data on epigenetic regulation of t-PA in human brain-derived cells. We demonstrate that treatment of cultured human neurons and human astrocytes with the histone deacetylase inhibitors trichostatin A (TSA) and MS-275 resulted in a two- to threefold increase in t-PA mRNA and protein expression levels. Next, we performed a chromatin immunoprecipitation assay on treated astrocytes with antibodies directed against acetylated histones H3 and H4 (both markers of gene activation). Treatment with MS-275 and TSA for 24 hours resulted in a significant increase in H3 acetylation, which could explain the observed increase in t-PA gene activity after the inhibition of histone deacetylation. Furthermore, DNA methylation analysis of cultured human neurons and astrocytes, as well as human postmortem brain tissue, revealed a stretch of unmethylated CpG dinucleotides in the proximal t-PA promoter, whereas more upstream CpGs were highly methylated. Taken together, these results implicate involvement of epigenetic mechanisms in the regulation of t-PA expression in the human brain. Less

Autophagy, a type II programmed cell death, is essential for cell survival under stress, e.g. lung injury, and bone marrow-derived mesenchymal stem cells (BM-MSCs) have g... More

Autophagy, a type II programmed cell death, is essential for cell survival under stress, e.g. lung injury, and bone marrow-derived mesenchymal stem cells (BM-MSCs) have great potential for cell therapy. However, the mechanisms underlying the BM-MSC activation of autophagy to provide a therapeutic effect in ischaemia/reperfusion-induced lung injury (IRI) remain unclear. Thus, we investigate the activation of autophagy in IRI following transplantation with BM-MSCs. Seventy mice were pre-treated with BM-MSCs before they underwent lung IRI surgery in vivo. Human pulmonary micro-vascular endothelial cells (HPMVECs) were pre-conditioned with BM-MSCs by oxygen-glucose deprivation/reoxygenation (OGD) in vitro. Expression markers for autophagy and the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signalling pathway were analysed. In IRI-treated mice, administration of BM-MSCs significantly attenuated lung injury and inflammation, and increased the level of autophagy. In OGD-treated HPMVECs, co-culture with BM-MSCs attenuated endothelial permeability by decreasing the level of cell death and enhanced autophagic activation. Moreover, administration of BM-MSCs decreased the level of PI3K class I and p-Akt while the expression of PI3K class III was increased. Finally, BM-MSCs-induced autophagic activity was prevented using the inhibitor LY294002. Administration of BM-MSCs attenuated lung injury by improving the autophagy level via the PI3K/Akt signalling pathway. These findings provide further understanding of the mechanisms related to BM-MSCs and will help to develop new cell-based therapeutic strategies in lung injury. Less

Reconstruction of the bladder by means of both natural and synthetic materials remains a challenge due to severe adverse effects such as mechanical failure. Here we inves... More

Reconstruction of the bladder by means of both natural and synthetic materials remains a challenge due to severe adverse effects such as mechanical failure. Here we investigate the application of spider major ampullate gland-derived dragline silk from the Nephila edulis spider, a natural biomaterial with outstanding mechanical properties and a slow degradation rate, as a potential scaffold for bladder reconstruction by studying the cellular response of primary bladder cells to this biomaterial. We demonstrate that spider silk without any additional biological coating supports adhesion and growth of primary human urothelial cells (HUCs), which are multipotent bladder cells able to differentiate into the various epithelial layers of the bladder. HUCs cultured on spider silk did not show significant changes in the expression of various epithelial-to-mesenchymal transition and fibrosis associated genes, and demonstrated only slight reduction in the expression of adhesion and cellular differentiation genes. Furthermore, flow cytometric analysis showed that most of the silk-exposed HUCs maintain an undifferentiated immunophenotype. These results demonstrate that spider silk from the Nephila edulis spider supports adhesion, survival and growth of HUCs without significantly altering their cellular properties making this type of material a suitable candidate for being tested in pre-clinical models for bladder reconstruction. Less

Background: Electronic cigarettes (EC) deliver aerosol by heating fluid containing nicotine. Cartomizer EC combine the fluid chamber and heating element in a single unit.... More

Background: Electronic cigarettes (EC) deliver aerosol by heating fluid containing nicotine. Cartomizer EC combine the fluid chamber and heating element in a single unit. Because EC do not burn tobacco, they may be safer than conventional cigarettes. Their use is rapidly increasing worldwide with little prior testing of their aerosol. Objectives: We tested the hypothesis that EC aerosol contains metals derived from various components in EC. Methods: Cartomizer contents and aerosols were analyzed using light and electron microscopy, cytotoxicity testing, x-ray microanalysis, particle counting, and inductively coupled plasma optical emission spectrometry. Results: The filament, a nickel-chromium wire, was coupled to a thicker copper wire coated with silver. The silver coating was sometimes missing. Four tin solder joints attached the wires to each other and coupled the copper/silver wire to the air tube and mouthpiece. All cartomizers had evidence of use before packaging (burn spots on the fibers and electrophoretic movement of fluid in the fibers). Fibers in two cartomizers had green deposits that contained copper. Centrifugation of the fibers produced large pellets containing tin. Tin particles and tin whiskers were identified in cartridge fluid and outer fibers. Cartomizer fluid with tin particles was cytotoxic in assays using human pulmonary fibroblasts. The aerosol contained particles >1 µm comprised of tin, silver, iron, nickel, aluminum, and silicate and nanoparticles (<100 nm) of tin, chromium and nickel. The concentrations of nine of eleven elements in EC aerosol were higher than or equal to the corresponding concentrations in conventional cigarette smoke. Many of the elements identified in EC aerosol are known to cause respiratory distress and disease. Conclusions: The presence of metal and silicate particles in cartomizer aerosol demonstrates the need for improved quality control in EC design and manufacture and studies on how EC aerosol impacts the health of users and bystanders. Less