Browsing by Subject "extracellular matrix"
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Item Activated human astrocyte-derived extracellular vesicles modulate neuronal uptake, differentiation and firing(Informa UK Limited, trading as Taylor & Francis Group, 2019-12-26) You, Yang; Borgmann, Kathleen; Edara, Venkata Viswanadh; Stacy, Satomi; Ghorpade, Anuja; Ikezu, TsuneyaAstrocytes in the central nervous system (CNS) provide supportive neural functions and mediate inflammatory responses from microglia. Increasing evidence supports their critical roles in regulating brain homoeostasis in response to pro-inflammatory factors such as cytokines and pathogen/damage-associated molecular pattern molecules in infectious and neurodegenerative diseases. However, the underlying mechanisms of the trans-cellular communication are still unclear. Extracellular vesicles (EVs) can transfer a large diversity of molecules such as lipids, nucleic acids and proteins for cellular communications. The purpose of this study is to characterize the EVs cargo proteins derived from human primary astrocytes (ADEVs) under both physiological and pathophysiological conditions. ADEVs were isolated from human primary astrocytes after vehicle (CTL) or interleukin-1beta (IL-1beta) pre-treatment. Label-free quantitative proteomic profiling revealed a notable up-regulation of proteins including actin-associated molecules, integrins and major histocompatibility complex in IL-1beta-ADEVs compared to CTL-ADEVs, which were involved in cellular metabolism and organization, cellular communication and inflammatory response. When fluorescently labelled ADEVs were added into primary cultured mouse cortical neurons, we found a significantly increased neuronal uptake of IL-1beta-ADEVs compared to CTL-ADEVs. We further confirmed it is likely due to the enrichment of surface proteins in IL-1beta-ADEVs, as IL-1beta-ADEVs uptake by neurons was partially suppressed by a specific integrin inhibitor. Additionally, treatment of neurons with IL-1beta-ADEVs also reduced neurite outgrowth, branching and neuronal firing. These findings provide insight for the molecular mechanism of the ADEVs' effects on neural uptake, neural differentiation and maturation, and its alteration in inflammatory conditions.Item Arginine Supplementation Promotes Extracellular Matrix and Metabolic Changes in Keratoconus(MDPI, 2021-08-13) McKay, Tina B.; Priyadarsini, Shrestha; Rowsey, Tyler; Karamichos, DimitriosKeratoconus (KC) is a common corneal ectatic disease that affects 1:500-1:2000 people worldwide and is associated with a progressive thinning of the corneal stroma that may lead to severe astigmatism and visual deficits. Riboflavin-mediated collagen crosslinking currently remains the only approved treatment to halt progressive corneal thinning associated with KC by improving the biomechanical properties of the stroma. Treatments designed to increase collagen deposition by resident corneal stromal keratocytes remain elusive. In this study, we evaluated the effects of arginine supplementation on steady-state levels of arginine and arginine-related metabolites (e.g., ornithine, proline, hydroxyproline, spermidine, and putrescine) and collagen protein expression by primary human corneal fibroblasts isolated from KC and non-KC (healthy) corneas and cultured in an established 3D in vitro model. We identified lower cytoplasmic arginine and spermidine levels in KC-derived constructs compared to healthy controls, which corresponded with overall higher gene expression of arginase. Arginine supplementation led to a robust increase in cytoplasmic arginine, ornithine, and spermidine levels in controls only and a significant increase in collagen type I secretion in KC-derived constructs. Further studies evaluating safety and efficacy of arginine supplementation are required to elucidate the potential therapeutic applications of modulating collagen deposition in the context of KC.Item BIOCHEMICAL AND HISTOLOGICAL CHARACTERIZATION OF TMJ(2014-03) Belmares, Ricardo; Lerma, Tanya; Guttmann, Geoffret; Taylor, Victor W.Objective of this project is to determine the composition of the the temporal mandibular joint (TMJ) from dissected human cadaver that will be essential for the development of stem cell therapy for individuals with diseased TMJ. This study aims to obtain a better understanding of the components that surround the cells in the TMJ. The components being studied include: collagen, elastin, and sulfated glycosaminoglycans. These molecules surround the cells forming a matrix that hold the TMJ intact. Understanding the exact composition of the TMJ matrix will is key to developing stem cell therapy for TMJ. Methods: Biochemical: Human TMJ discs and attachments were dissected from recently deceased bodies and analyzed both histologically and biochemically. Samples dry weight obtained. Follow up studies include: measuring DNA content, collagen, and sulfated glycosaminoglycans. All these will be done using commercially available kits. Histological: Histological preparations were made with various stains: Alcian Blue for assay acidic glycans; Verhoeff’s Elastic (VEG); and Hematoxylin Eosin (H&E). Results: Elastin composition of human samples show differences compared to previous pig studies. TMJ disc has a denser matrix with less elastic fibers than the TMJ attachments. TMJ attachments have more visible nuclei than disc with H&E staining. TMJ complex is saturated with sulfated GAGs. Conclusion: This study shows that matrix composition of human TMJ may differ from previous pig studies. Further studies from human samples using various ages can better elucidate the matrix composition for targeted human TMJ stem cell therapy. Purpose (a): Objective: Characterizing biochemical and histological composition of the human temporal mandibular joint (TMJ) disc is essential to development of stem cell therapy for diseased TMJ. Components of extracellular matrix (ECM) analyzed are elastin, collagen and sulfated glycosaminoglycan (GAG). Methods (b): Biochemical: Human TMJ discs were dissected from recently deceased bodies and analyzed histologically and biochemically. Five regions of the disc and six distal attachments examined. Following dissection, wet weights recorded. Samples lyophilized to obtain dry weight. Dried samples were digested in a 125mg/mL papain solution overnight at 600C. Follow up studies include: DNA content (measured with the Quant-iT Picrogreen dsDNA Assay Kit (Invitrogen). Following hydrolysis with 4N NaOH for 20 minutes at 110 0C, collagen content to be quantified with modified chloramine-T hydroxyproline assay. Sulfated GAG content will be quantified using Blyscan Glycosaminoglycan Assay Kit (Accurate Chemical and Scientific Corp.). N= 6 samples per group for all biochemical analysis. Histological: Histological preparations were made with various stains: Alcian Blue for assay acidic glycans; Verhoeff’s Elastic (VEG); and Hematoxylin Eosin (H&E). Results (c): Elastin composition of human samples show differences compared to previous porcine studies. TMJ disc has a denser matrix with less elastic fibers than distal attachments. TMJ attachments have more visible nuclei than disc with H&E staining. TMJ complex is saturated with sulfated GAGs. Conclusions (d): This study shows that ECM composition of human TMJ may differ from previous porcine studies. Further studies from human samples using various ages can further elucidate the ECM composition for targeted human TMJ stem cell therapy.Item Expression of Procollagen C Proteinase Enhancer Proteins in Trabecular Cells and Tissues(2013-05-01) Naik, Monal; Robert WordingerPrimary open angle glaucoma (POAG) is the most common form of glaucoma. Ocular hypertension is a major risk factor for POAG and is caused by increased aqueous humor (AH) outflow resistance in the trabecular meshwork (TM). Increased extracellular matrix (ECM) deposition within the TM is correlated with ocular hypertension. Transforming Growth Factor beta 2 (TGFb2) levels are elevated in the AH and TM of POAG patients, and TGFb2 increases ECM protein expression, aqueous outflow resistance, and intraocular pressure (IOP). Recently, TGFβ2 was found to induce bone morphogenetic protein 1 (BMP1) expression in TM cells suggesting that BMP1 activity might be involved in glaucoma pathogenesis. Procollagen C proteinase enhancers (PCOLCE 1 and PCOLCE 2) regulate BMP1 activity. Therefore, PCOLCE1 and PCOLCE2 may play an important role(s) in regulating ECM structural changes in the TM, and contribute to AH outflow resistance and elevated IOP in glaucoma. The purpose of this study was to determine if human TM cells and tissues express PCOLCE1 and PCOLCE2 and whether TGFb2 induces their expression. This is the first documentation that PCOLCE1 and PCOLCE2 are expressed in TM cells and tissues and that TGFb2 does induce expression of PCOLCE1.Item Extracellular PACE4 is increased following transient oxygen glucose deprivation in Optic Nerve Astrocytes(2008-05-01) Fuller, John Anthony; Wordinger, Robert J.; Clark, Abbot F.; Krishnamoorthy, Raghu R.Fuller, John Anthony Extracellular PACE4 is increased following transient oxygen glucose deprivation in Optic Nerve Astrocytes. Doctor of Philosophy (Biomedical Sciences), May, 2008, 140 pp., 2 tables, 25 illustrations, bibliography, 218 titles. Primary Open Angle Glaucoma (POAG) is a family of heterogeneous optic neuropathies characterized by progressive retinal ganglion cell (RGC) death that leads to peripheral vision loss and eventually blindness. Various risk factors are associated with glaucoma, however the molecular mechanisms leading to RGC cell death remain unknown. The optic nerve serves as the conduit for the transmission of retinal ganglion action potentials to the brain. The cells that compromise the optic nerve form a scaffold that forms a physical support for the RGC axons. One cell type found throughout the optic nerve and associated with the RGC axon is the optic nerve astrocyte (ONA). Astrocytes are a predominant cell throughout the CNS and are believed to play crucial roles in metabolic, growth factor, and structural support, and respond to protect neurons during injury. The neuronal-glial interface in the optic nerve is poorly understood and believed to plan an important role in POAG pathophysiology, as unmyelenated RGC axons have direct contact with astrocyte processes. IN this study, the subtilisin-like Proprotein Convertases, (SPC) a family of proteases responsible for cleaving a wide variety of protein substrates, were examined in the retina and optic nerve head. PACE4, an SPC found to be secreted and active in the extracellular matrix was found to be highly expressed in the optic nerve, and colocalized to Mϋller cells in the retina and astrocytes in the optic nerve. Exposure of primary optic nerve astrocytes to oxygen-glucose deprivation (OGD) induces an increase in PACE4 mRNA. Furthermore, protein levels of extracellular, processed PACE4 increase following transient ODG, whereas the pro form of the molecule is degraded, and is believed to be chaperoned by the cleaved cysteine rich domain, a product found at high levels in the optic nerve in situ and the ONA in vitro. Due to the extracellular activity of PACE4, we hypothesized that it may regulate the bioactivity of TGF-β2, a growth factor believed to be involved in glaucoma-associated ONH remodeling by inducing the production of extracellular matrix (ECM). When PACE4 is inhibited via siRNA-mediated knockdown, as well as extracellular inactivation, TGF-β2 levels decrease. In addition, fibronectin, a major component of the ECM, is decreased. Furthermore, there is an increase in latent TGF-β2 secreted from the cell. It is therefore possible that PACE4 plays an active role in extracellular growth factor maturation, and may be a central mediator for growth factor bioactivity in the glaucomatous ONA.Item miRNA Profiling of Human Optic Nerve Head Astrocytes Exposed to Cyclic Stretch(2021-05) Rangan, Rajiv S.; Tovar-Vidales, Tara; Clark, Abbot F.; Liu, YangGlaucoma is a leading cause of irreversible blindness. Vision loss results from the degeneration and death of retinal ganglion cells (RGCs) and their axons. The primary risk factor for glaucoma is increased intraocular pressure (IOP) (2). Elevated IOP results in aberrations in the biomechanical properties of ocular tissues - including the transmission of biomechanical stretch through the reticulated, fibroelastic region of the optic nerve head (ONH) known as the lamina cribrosa (LC) (6). Cells of the LC are sensitive to biomechanical stretch and respond to increased stretch and pressure to promote the excessive synthesis of extracellular matrix (ECM) proteins and ECM remodeling (15,17). These responses promote a fibrotic environment within the LC that can cause mechanical damage to the axons of RGCs. ONH astrocytes represent one of the major cell types of the LC and are believed to contribute significantly to pathological ECM remodeling at the LC during glaucoma (11). ONH astrocytes also demonstrate a dysregulated pattern of protein expression when exposed to stretch (17). The mechanism that underlies this stretch-induced, aberrant dysregulation is unknown. MicroRNA (miRNA) dysregulation may represent one of the mechanisms contributing to the differential protein expression patterns seen in ONH astrocytes exposed to stretch. In this study we examine the miRNA profiles of ONH astrocytes exposed to cyclic stretch.Item Role of Extracellular Matrix Crosslinking Enzyme Tissue Transglutaminase in Trabecular Meshwork Homeostasis and Regulating Intraocular Pressure(2017-12-01) Raychaudhuri, Urmimala; Clark, Abbot F.; Krishnamoorthy, Raghu R.; Mao, WeimingIncreased intraocular pressure (IOP) is one of the major known risk factors for primary open angle glaucoma (POAG). The main cause of IOP elevation is obstruction to aqueous humor (AH) outflow at the trabecular meshwork (TM) in the eye. Cellularity and ECM turnover rates affect the normal physiology of the TM tissue. Various factors including transforming growth factor (TGFβ2) have been found responsible for a large number of glaucomatous changes. Increased expression and activity of tissue transglutaminase (TGM2), an enzyme induced by TGFβ2, has been seen in the glaucomatous TM. TGM2 can covalently crosslink ECM proteins including collagens, fibronectin and elastin. The purpose of this study was to investigate the role of TGM2 in ocular hypertension. For this, we overexpressed TGM2 using adenovirus serotype 5 (Ad5), which has tropism for TM cells, in TM cells in vitro and in mouse eyes in vivo. In vitro, we validated overexpression of TGM2 using Ad5.TGM2 and also found increased crosslinking following overexpression. For our animal studies we used BALB/cJ and C57BL/6J mice. In our mouse models, following intravitreal injection of Ad5.TGM2, we saw a significant increase in IOP and decrease in AH outflow facility in TGM2 overexpressed eyes compared to contralateral eyes. Immunohistochemical staining showed that there was increased expression of TGM2 and increased crosslinking in the TM region. There also appeared to be increased fibronectin at the TM region. We followed this with a knockout (KO) study to determine whether TGM2 KO could affect IOP. For this we used TGM2 floxed mice. Following intravitreal injection of Ad5.Cre, we find a significant reduction in IOP. We also found that KO of TGM2 significantly reduced TGFβ2 induced ocular hypertension. We further tested a small molecule TGM2 inhibitor ZM 449829. This inhibitor binds to TGM2 and inhibits crosslinking activity by locking it in an inactive state. In our findings, we observed that when treated with 5nM ZM 449829, at 48 hours it inhibited TGM2 crosslinking in 3 primary human glaucomatous cell strains. It also appears to reduce fibronectin deposition. We performed some preliminary tests of this drug in vivo. 5μM of the drug dissolved in PBS was administered as eye drops as once daily dosing. At 3 weeks, gross morphology of the eye and cornea looked normal. IOP measurements taken once a week till 3 weeks also did not show any aberrant changes. Overall, our findings suggest that TGM2 plays a significant role in inducing ocular hypertension. This makes TGM2 a potential therapeutic target; therefore inhibition or amelioration of TGM2 crosslinking activity such as with potent inhibitors like ZM 449829 should be further studied as a novel therapeutic strategy for glaucoma.