The ocular surface comprises the wet mucosal epithelia of the cornea and conjunctiva, the associated glands, and the overlying tear film. Epitheliopathy is the common pathologic outcome when the ...ocular surface is subjected to oxidative stress. Whether different stresses act via the same or different mechanisms is not known. Dynasore and dyngo-4a, small molecules developed to inhibit the GTPase activity of classic dynamins DNM1, DNM2, and DNM3, but not mdivi-1, a specific inhibitor of DNM1L, protect corneal epithelial cells exposed to the oxidant tert-butyl hydroperoxide (tBHP). Here we report that, while dyngo-4a is the more potent inhibitor of endocytosis, dynasore is the better cytoprotectant. Dynasore also protects corneal epithelial cells against exposure to high salt in an in vitro model of dysfunctional tears in dry eye. We now validate this finding in vivo, demonstrating that dynasore protects against epitheliopathy in a mouse model of dry eye. Knockdown of classic dynamin DNM2 was also cytoprotective against tBHP exposure, suggesting that dynasore's effect is at least partially on target. Like tBHP and high salt, exposure of corneal epithelial cells to nitrogen mustard upregulated the unfolded protein response and inflammatory markers, but dynasore did not protect against nitrogen mustard exposure. In contrast, mdivi-1 was cytoprotective. Interestingly, mdivi-1 did not inhibit the nitrogen mustard-induced expression of inflammatory cytokines. We conclude that exposure to tBHP or nitrogen mustard, two different oxidative stress agents, cause corneal epitheliopathy via different pathologic pathways. SIGNIFICANCE STATEMENT: Results presented in this paper, for the first time, implicate the dynamin DNM2 in ocular surface epitheliopathy. The findings suggest that dynasore could serve as a new topical treatment for dry eye epitheliopathy and that mdivi-1 could serve as a medical countermeasure for epitheliopathy due to nitrogen mustard exposure, with potentially increased efficacy when combined with anti-inflammatory agents and/or UPR modulators.
Dysregulation of calcium homeostasis can precipitate a cascade of pathological events that lead to tissue damage and cell death. Dynasore is a small molecule that inhibits endocytosis by targeting ...classic dynamins. In a previous study, we showed that dynasore can protect human corneal epithelial cells from damage due to tert-butyl hydroperoxide (tBHP) exposure by restoring cellular calcium (Ca2+) homeostasis. Here we report results of a follow-up study aimed at identifying the source of the damaging Ca2+. Store-operated Ca2+ entry (SOCE) is a cellular mechanism to restore intracellular calcium stores from the extracellular milieu. We found that dynasore effectively blocks SOCE in cells treated with thapsigargin (TG), a small molecule that inhibits pumping of Ca2+ into the endoplasmic reticulum (ER). Unlike dynasore however, SOCE inhibitor YM-58483 did not interfere with the cytosolic Ca2+ overload caused by tBHP exposure. We also found that dynasore effectively blocks Ca2+ release from internal sources. The inefficacy of inhibitors of ER Ca2+ channels suggested that this compartment was not the source of the Ca2+ surge caused by tBHP exposure. However, using a Ca2+-measuring organelle-entrapped protein indicator (CEPIA) reporter targeted to mitochondria, we found that dynasore can block mitochondrial Ca2+ release due to tBHP exposure. Our results suggest that dynasore exerts multiple effects on cellular Ca2+ homeostasis, with inhibition of mitochondrial Ca2+ release playing a key role in protection of corneal epithelial cells against oxidative stress due to tBHP exposure.Dysregulation of calcium homeostasis can precipitate a cascade of pathological events that lead to tissue damage and cell death. Dynasore is a small molecule that inhibits endocytosis by targeting classic dynamins. In a previous study, we showed that dynasore can protect human corneal epithelial cells from damage due to tert-butyl hydroperoxide (tBHP) exposure by restoring cellular calcium (Ca2+) homeostasis. Here we report results of a follow-up study aimed at identifying the source of the damaging Ca2+. Store-operated Ca2+ entry (SOCE) is a cellular mechanism to restore intracellular calcium stores from the extracellular milieu. We found that dynasore effectively blocks SOCE in cells treated with thapsigargin (TG), a small molecule that inhibits pumping of Ca2+ into the endoplasmic reticulum (ER). Unlike dynasore however, SOCE inhibitor YM-58483 did not interfere with the cytosolic Ca2+ overload caused by tBHP exposure. We also found that dynasore effectively blocks Ca2+ release from internal sources. The inefficacy of inhibitors of ER Ca2+ channels suggested that this compartment was not the source of the Ca2+ surge caused by tBHP exposure. However, using a Ca2+-measuring organelle-entrapped protein indicator (CEPIA) reporter targeted to mitochondria, we found that dynasore can block mitochondrial Ca2+ release due to tBHP exposure. Our results suggest that dynasore exerts multiple effects on cellular Ca2+ homeostasis, with inhibition of mitochondrial Ca2+ release playing a key role in protection of corneal epithelial cells against oxidative stress due to tBHP exposure.
Epitheliopathy at the ocular surface is a defining sign of dry eye disease, a common disorder that affects 10% to 30% of the world's population. Hyperosmolarity of the tear film is one of the main ...drivers of pathology, with subsequent endoplasmic reticulum (ER) stress, the resulting unfolded protein response (UPR), and caspase-3 activation implicated in the pathway to programmed cell death. Dynasore, is a small molecule inhibitor of dynamin GTPases that has shown therapeutic effects in a variety of disease models involving oxidative stress. Recently we showed that dynasore protects corneal epithelial cells exposed to the oxidant tBHP, by selective reduction in expression of CHOP, a marker of the UPR PERK branch. Here we investigated the capacity of dynasore to protect corneal epithelial cells subjected to hyperosmotic stress (HOS). Similar to dynasore's capacity to protect against tBHP exposure, dynasore inhibits the cell death pathway triggered by HOS, protecting against ER stress and maintaining a homeostatic level of UPR activity. However, unlike with tBHP exposure, UPR activation due to HOS is independent of PERK and mostly driven by the UPR IRE1 branch. Our results demonstrate the role of the UPR in HOS-driven damage, and the potential of dynasore as a treatment to prevent dry eye epitheliopathy.
MicroRNA (miRNA) has shown to enhance or inhibit cell proliferation, differentiation and activity of different cell types in bone tissue. The discovery of miRNA actions and their targets has helped ...to identify them as novel regulations actors in bone. Various studies have shown that miRNA deregulation mediates the progression of bone-related pathologies, such as osteoporosis.
The present review intends to give an exhaustive overview of miRNAs with experimentally validated targets involved in bone homeostasis and highlight their possible role in osteoporosis development. Moreover, the review analyzes miRNAs identified in clinical trials and involved in osteoporosis.
•Review of miRNAs in osteogenic pathways•Review of miRNAs in osteoclastogenic pathways•Review of circulating miRNAs implicated in osteoporosis•Identification of circulating miRNAs as possible markers of osteoporosis
Proteostasis refers to all the processes that maintain the correct expression level, location, folding and turnover of proteins, essential to organismal survival. Both inside cells and in body ...fluids, molecular chaperones play key roles in maintaining proteostasis. In this article, we focus on clusterin, the first-recognized extracellular mammalian chaperone, and its role in diseases of the eye. Clusterin binds to and inhibits the aggregation of proteins that are misfolded due to mutations or stresses, clears these aggregating proteins from extracellular spaces, and facilitates their degradation. Clusterin exhibits three main homeostatic activities: proteostasis, cytoprotection, and anti-inflammation. The so-called "protein misfolding diseases” are caused by aggregation of misfolded proteins that accumulate pathologically as deposits in tissues; we discuss several such diseases that occur in the eye. Clusterin is typically found in these deposits, which is interpreted to mean that its capacity as a molecular chaperone to maintain proteostasis is overwhelmed in the disease state. Nevertheless, the role of clusterin in diseases involving such deposits needs to be better defined before therapeutic approaches can be entertained. A more straightforward case can be made for therapeutic use of clusterin based on its proteostatic role as a proteinase inhibitor, as well as its cytoprotective and anti-inflammatory properties. It is likely that clusterin works together in this way with other extracellular chaperones to protect the eye from disease, and we discuss several examples. We end this article by predicting future steps that may lead to development of clusterin as a biological drug.
•Clusterin was the first extracellular mammalian chaperone identified.•Clusterin has proteostatic, cytoprotective and anti-inflammatory activities.•Insufficient clusterin may result in failure to resolve extracellular deposits.•Clusterin's homeostatic activities suggest it as a biotherapeutic for eye disease.•Clusterin may work together with other molecular chaperones found in the eye.
We report a two-span, 67-km space-division-multiplexed (SDM) wavelength-division-multiplexed (WDM) system incorporating the first reconfigurable optical add-drop multiplexer (ROADM) supporting ...spatial superchannels and the first cladding-pumped multicore erbium-doped fiber amplifier directly spliced to multicore transmission fiber. The ROADM subsystem utilizes two conventional 1 × 20 wavelength selective switches (WSS) each configured to implement a 7 × (1 × 2) WSS. ROADM performance tests indicate that the subchannel insertion losses, attenuation accuracies, and passband widths are well matched to each other and show no significant penalty, compared to the conventional operating mode for the WSS. For 6 × 40 × 128-Gb/s SDM-WDM polarization-multiplexed quadrature phase-shift-keyed (PM-QPSK) transmission on 50 GHz spacing, optical signal-to-noise ratio penalties are less than 1.6 dB in Add, Drop, and Express paths. In addition, we demonstrate the feasibility of utilizing joint signal processing of subchannels in this two-span, ROADM system.
A new urea-containing metal–organic framework (MOF) was synthesized to act as a heterogeneous catalyst. Ureas are well-known for self-recognition and aggregation behavior, resulting in loss of ...catalytic competency. The catalyst spatial isolation achievable in a porous MOF environment suggests a potentially general solution. The combination of a symmetrical urea tetracarboxylate strut, 4,4′-bipyridine, and Zn(NO3)2·6H2O under solvothermal conditions afforded a new microporous MOF (NU-601). This material is indeed an effective hydrogen-bond-donor catalyst for Friedel–Crafts reactions between pyrroles and nitroalkenes, whereas a homogeneous urea is much less competent. The higher rates of reaction of small substrates relative to larger ones with NU-601 strongly suggest that catalysis primarily occurs within the pores of this new material rather than on its exterior. To the best of our knowledge, this approach is the first example of specific engineering of successful hydrogen-bonding catalysis into a MOF material.
Abstract Background Age-related macular degeneration (AMD) is a leading cause of visual loss among the elderly. A key cell type involved in AMD, the retinal pigment epithelium, expresses a G ...protein–coupled receptor that, in response to its ligand, L-DOPA, up-regulates pigment epithelia–derived factor, while down-regulating vascular endothelial growth factor. In this study we investigated the potential relationship between L-DOPA and AMD. Methods We used retrospective analysis to compare the incidence of AMD between patients taking vs not taking L-DOPA. We analyzed 2 separate cohorts of patients with extensive medical records from the Marshfield Clinic (approximately 17,000 and approximately 20,000) and the Truven MarketScan outpatient and databases (approximately 87 million) patients. We used International Classification of Diseases, 9th Revision codes to identify AMD diagnoses and L-DOPA prescriptions to determine the relative risk of developing AMD and age of onset with or without an L-DOPA prescription. Results In the retrospective analysis of patients without an L-DOPA prescription, AMD age of onset was 71.2, 71.3, and 71.3 in 3 independent retrospective cohorts. Age-related macular degeneration occurred significantly later in patients with an L-DOPA prescription, 79.4 in all cohorts. The odds ratio of developing AMD was also significantly negatively correlated by L-DOPA (odds ratio 0.78; confidence interval, 0.76-0.80; P <.001). Similar results were observed for neovascular AMD ( P <.001). Conclusions Exogenous L-DOPA was protective against AMD. L-DOPA is normally produced in pigmented tissues, such as the retinal pigment epithelium, as a byproduct of melanin synthesis by tyrosinase. GPR143 is the only known L-DOPA receptor; it is therefore plausible that GPR143 may be a fruitful target to combat this devastating disease.
In mammals, tissue damage is usually repaired by activation of a fibrotic response which saves the life of the organism, but which can never restore function to the damaged organ. In addition, ...fibrotic responses form the basis for diverse pathologies, including many that occur in the eye. It is intriguing, therefore, to observe the occasional circumstances in which repair in mammals appears to take on a regenerative character, such as during fetal wound healing or in certain types of corneal wounds. The thesis of this chapter is that the choice between regeneration or fibrosis lies in the control of fibroblast phenotype. The cornea of the eye has several features which make it a particularly useful model for the study of fibroblast phenotype. Studies discussed herein, identify failure to activate the transcription factor NF-κB as a control mechanism for inhibiting fibroblast activation in the cornea. Evidence is further presented for the view that transition in fibroblast phenotype in repair tissue is not simply a matter of differential gene expression, but is a developmental event which reflects changes in the hard wiring of signalling pathways by which the cell responds to environmental input.
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