INTRA-MITOCHONDRIAL INJURY DURING ISCHEMIA-REPERFUSION

Cardiac injury is increased following ischemia-reperfusion. Mitochondria are the “effector organelles” that are damaged during ischemia (ISC) when there is no blood flow. Resumption of metabolism by damaged mitochondria during reperfusion (REP) results in increased cell injury. Current therapeutic interventions to pre-condition and post-condition the heart during ISC are ineffective during certain conditions like aging and diabetes due to defects in the signaling cascades. In contrast, mitochondrial-based strategies are effective in protecting the heart during ISC-REP. Hence direct therapeutic targeting of dysfunctional mitochondria will provide the potential to bypass the upstream signaling defects and intervene directly upon the effector organelle. Novel mitochondrial-targeted therapy relies on understanding the sites in the electron transport chain (ETC) that are damaged by ISC and produce cell-injury during REP. This project identifies a novel pathological role of cytochrome c in depleting cardiolipin during ischemia after which the mitochondria are in a defective condition that leads to additional cell death during reperfusion. During ischemia oxidants from complex III oxidize cytochrome c, forming a peroxidase, which causes oxidative damage and depletion of cardiolipin. Depletion of cardiolipin disrupts normal physiology and augments cell death. Identification of the innovative pathobiology during ISC-REP recognizes a novel therapeutic target, cytochrome c peroxidase, which can be a focal point for new therapeutic interventions to decrease cardiac injury. In order to maintain homeostatis, living organisms have the methionine sulfoxide reductase system, which reduce both free and protein bound Met(O) back to methionine (Met) in the presence of thioredoxin. Oxidized Trx is inactive and unable to bind to ASK1 thereby activating ASK1 and causing cell death via p38/JNK pathways thereby contributing to the pathogenesis of myocardial ISC-REP injury. In this study we have shown that inhibition of ASK1 protects the heart during REP via the modulation of mitochondria that sustained damage during ISC. The mitochondrial-based mechanism of cardioprotection with ASK1 inhibition enhanced the functional integrity of the inner mitochondrial membrane retaining cytochrome c thereby decreasing cell death. This therapeutic intervention is a key step to achieve the ultimate goal to improve clinical outcomes in patients that suffer an acute myocardial infarction

PREPARATION AND CHARACTERIZATION OF STABLE MACROPOROUS TITANIA NANO-WELLS

Silica based sol-gel thin films have been extensively studied because of their advantages that include optical transparency, high biodegradability and low intrinsic fluorescence. However, one concern with silica based sol-gel derived materials is their long term stability in aqueous solutions. Another concern is their limited porosity. These two concerns limit the application of these materials in catalysis and separations. The main objective of this study is to prepare porous, thin films using titanium alkoxides as precursors and evaluate their long term stability in aqueous solutions. Colloidal crystal templating will be used to introduce macrosized pores into the titania network. The materials will be characterized using SEM and AFM. To prove that the templated films provide access to the underlying surface of the electrode, a conducting metal like copper was electrodeposited inside the nano-wells. The stability of titania and silica films will be evaluated over a two month period using cyclic voltammetry with three redox probes

Photothermal Deoxygenation of Graphite Oxide with Laser Excitation in Solution and Graphene-Aided Increase in Water Temperature

We report the development of a facile laser reduction method for the synthesis of laser converted graphene (LCG) from graphite oxide (GO). The method provides a solution processable synthesis of individual graphene sheets in water under ambient conditions without the use of any chemical reducing agent. We also report on the high performance of GO and LCG for the efficient conversion of the laser radiation into usable heat, particularly for heating water for a variety of potential thermal, thermochemical, and thermomechanical applications

Role of Matrix Metalloproteinases 2 and 9 in Lacrimal Gland Disease in Animal Models of Sjögren's Syndrome

Citation: Aluri HS, Kublin CL, Thotakura S, et al. Role of matrix metalloproteinases 2 and 9 in lacrimal gland disease in animal models of Sjögren's syndrome. Invest Ophthalmol Vis Sci. 2015;56:5218-5228. DOI:10.1167/ iovs.15-17003 PURPOSE. Chronic inflammation of the lacrimal gland results in changes in the composition of the extracellular matrix (ECM), which is believed to compromise tissue repair. We hypothesized that increased production/activity of matrix metalloproteinases (MMPs), especially MMP-2 and -9, in inflamed lacrimal glands modifies the ECM environment, therefore disrupting tissue repair. METHODS. The lacrimal glands from female MRL/lpr and male NOD mice along with their respective control strains were harvested and divided into three pieces and processed for histology, immunohistochemistry, zymography, Western blotting, and RNA analyses. In another study, MRL/lpr mice were treated for 5 weeks with a selective MMP2/9 inhibitor peptide or a control peptide. At the end of treatment, the lacrimal glands were excised and the tissue was processed as described above. RESULTS. There was a 2.5-and 2.7-fold increase in MMP2 gene expression levels in MRL/lpr and NOD mice, respectively. Matrix metalloproteinase 2 and 9 enzymatic activities and protein expression levels were significantly upregulated in the lacrimal glands of MRL/lpr and NOD mice compared to controls. Treatment with the MMP2/9 inhibitor resulted in decreased activity of MMP-2 and -9 both in vitro and in vivo. Importantly, MMP2/9 inhibitor treatment of MRL/lpr mice improved aqueous tear production and resulted in reduced number and size of lymphocytic foci in diseased lacrimal glands. CONCLUSIONS. We conclude that MMP2/9 expression and activity are elevated in lacrimal glands of two murine models of Sjögren's syndrome, suggesting that manipulation of MMP2/9 activity might be a potential therapeutic target in chronically inflamed lacrimal glands

RNA-Seq and CyTOF immuno-profiling of regenerating lacrimal glands identifies a novel subset of cells expressing muscle-related proteins

<div><p>The purpose of the present studies was to use CyTOF and RNA-Seq technologies to identify cells and genes involved in lacrimal gland repair that could be targeted to treat diseases of lacrimal gland dysfunction. Lacrimal glands of female BALB/c mice were experimentally injured by intra-glandular injection of interleukin 1 alpha (IL-1α). The lacrimal glands were harvested at various time points following injury (1 to 14 days) and used to either prepare single cell suspensions for CyTOF immuno-phenotyping analyses or to extract RNA for gene expression studies using RNA-Seq. CyTOF immuno-phenotyping identified monocytes and neutrophils as the major infiltrating populations 1 and 2 days post injury. Clustering of significantly differentially expressed genes identified 13 distinct molecular signatures: 3 associated with immune/inflammatory processes included genes up-regulated at days 1–2 and 3 associated with reparative processes with genes up-regulated primarily between days 4 and 5. Finally, clustering identified 65 genes which were specifically up-regulated 2 days post injury which was enriched for muscle specific genes. The expression of select muscle-related proteins was confirmed by immunohistochemistry which identified a subset of cells expressing these proteins. Double staining experiments showed that these cells are distinct from the myoepithelial cells. We conclude that experimentally induced injury to the lacrimal gland leads to massive infiltration by neutrophils and monocytes which resolved after 3 days. RNAseq and immunohistochemistry identified a group of cells, other than myoepithelial cells, that express muscle-related proteins that could play an important role in lacrimal gland repair.</p></div

Quantification of immune cell populations.

<p>The number of cells per thousand of analyzed cells were plotted by day for (A) CD45+ (immune cells) CD45- (non-immune cells), (B) cells with large population change, monocytes and neutrophils, and (C) cells with small population change, natural killer (NK), B, plasmacytoid dendritic (pDC), CD4+ and CD8+ T cells. Insert shows representative immunofluorescence staining of Ly-6G (neutrophils) 1 day post IL-1α injection.</p

Venn diagram of overlap in significant gene regulation for each day.

<p>A Venn diagram of the differentially expressed genes at days 1, 2, 3, 4, and 5 was created to identify the overlap in gene expression.</p

Delivery of Bone Marrow-Derived Mesenchymal Stem Cells Improves Tear Production in a Mouse Model of Sjögren’s Syndrome

The purpose of the present study was to test the potential of mouse bone marrow-derived mesenchymal stem cells (BD-MSCs) in improving tear production in a mouse model of Sjögren’s syndrome dry eye and to investigate the underlying mechanisms involved. NOD mice (n=20) were randomized to receive i.p. injection of sterile phosphate buffered saline (PBS, control) or murine BD-MSCs (1 × 106 cells). Tears production was measured at baseline and once a week after treatment using phenol red impregnated threads. Cathepsin S activity in the tears was measured at the end of treatment. After 4 weeks, animals were sacrificed and the lacrimal glands were excised and processed for histopathology, immunohistochemistry, and RNA analysis. Following BD-MSC injection, tears production increased over time when compared to both baseline and PBS injected mice. Although the number of lymphocytic foci in the lacrimal glands of treated animals did not change, the size of the foci decreased by 40.5% when compared to control animals. The mRNA level of the water channel aquaporin 5 was significantly increased following delivery of BD-MSCs. We conclude that treatment with BD-MSCs increases tear production in the NOD mouse model of Sjögren’s syndrome. This is likely due to decreased inflammation and increased expression of aquaporin 5

Representative molecular signatures of lacrimal gland inflammation and repair.

<p>Expression (log2[fold change]) pattern of clusters representative of the 8 consolidated clusters were plotted for days 1, 2, 3, 4, 5, and 7. Threshold for significant up/down-regulation (+/- 1 = log2[+/-2]) is indicated by dotted blue line.</p