Analysis of sphingolipids in human corneal fibroblasts from normal and keratoconus patients

Qi, Hui; Priyadarsini, Shrestha; Nicholas, Sarah E.; Sarker-Nag, Akhee; Allegood, Jeremy C.; Chalfant, Charles E.; Mandal, Nawajes A.; Karamichos, Dimitrios

doi:10.1194/jlr.m067264

Cited by 20 publications

(18 citation statements)

References 68 publications

(94 reference statements)

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“…As an example, phosphatidylserine (PS), before being present in the membrane inner leaflet, is externalized [65], and cholesterol synthesis increases [27,66,67]. These changes result in a negative net charge on the cancer cell membrane, contrary to a neutral surface charge found on non-cancerous cell membranes [68][69][70].…”

Section: Discussionmentioning

confidence: 99%

Pisum sativum Defensin 1 Eradicates Mouse Metastatic Lung Nodules from B16F10 Melanoma Cells

Amaral

Santos

Andrade

et al. 2020

IJMS

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Psd1 is a pea plant defensin which can be actively expressed in Pichia pastoris and shows broad antifungal activity. This activity is dependent on fungal membrane glucosylceramide (GlcCer), which is also important for its internalization, nuclear localization, and endoreduplication. Certain cancer cells present a lipid metabolism imbalance resulting in the overexpression of GlcCer in their membrane. In this work, in vitroassays using B16F10 cells showed that labeled fluorescein isothiocyanate FITC-Psd1 internalized into live cultured cells and targeted the nucleus, which underwent fragmentation, exhibiting approximately 60% of cells in the sub-G0/G1 stage. This phenomenon was dependent on GlcCer, and the participation of cyclin-F was suggested. In a murine lung metastatic melanoma model, intravenous injection of Psd1 together with B16F10 cells drastically reduced the number of nodules at concentrations above 0.5 mg/kg. Additionally, the administration of 1 mg/kg Psd1 decreased the number of lung inflammatory cells to near zero without weight loss, unlike animals that received melanoma cells only. It is worth noting that 1 mg/kg Psd1 alone did not provoke inflammation in lung tissue or weight or vital signal losses over 21 days, inferring no whole animal cytotoxicity. These results suggest that Psd1 could be a promising prototype for human lung anti-metastatic melanoma therapy.

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Section: Discussionmentioning

confidence: 99%

Pisum sativum Defensin 1 Eradicates Mouse Metastatic Lung Nodules from B16F10 Melanoma Cells

Amaral

Santos

Andrade

et al. 2020

IJMS

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“…The chain length of Cer depends on the activity of the specific CerS. CerS1 synthesizes mainly C18-Cer; CerS4 synthesizes C18-/C20-/C24-Cer; CerS5 and CerS6 synthesize mostly C14-/C16-/C18-Cer; CerS2 synthesizes preferentially C22-/C24-Cer; and CerS3 synthesizes very long-chain Cers (>C26-Cer) (36,(56)(57)(58)(59). Our data may suggest an activation of CerS2 and an inhibition of CerS3.…”

Section: Discussionmentioning

confidence: 69%

“…Sphingolipids were analyzed in the Lipidomics Core facility at Virginia Commonwealth University, Richmond, VA following previously established procedures (36). All internal standards were procured from Avanti Polar Lipids and added to samples at a ratio of ethanol:methanol:water (7:2:1) as a cocktail of 500 pmol each.…”

Section: Quantitative Analysis Of Sphingolipidsmentioning

confidence: 99%

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Overexpression of acid ceramidase (ASAH1) protects retinal cells (ARPE19) from oxidative stress

Sugano

Edwards

Saha

et al. 2019

Journal of Lipid Research

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Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly population. More than 80% of AMD cases are of the dry-AMD classification, which characteristically features geographic atrophy resulting from the loss of retinal pigment epithelial (RPE) cells (1, 2). Dry-AMD often converts to wet-AMD, which is visionthreatening. Accumulation of undigested cellular debris (e.g., drusen), oxidative stress, and subsequent death and degeneration of RPE cells are important contributors to AMD pathology (3,4). Accumulating evidence suggests that the cellular sphingolipid mediator, ceramide (Cer), which acts as a second messenger to induce apoptosis, might play a role in RPE cell death (5-7). Here, we investigate for the first time the role of Cer in human RPE-derived ARPE19 cells under oxidative stress by removing excess cellular Cer via genetic manipulation of Cer-hydrolyzing enzymes.In addition to being a critical component of membrane structure and function, Cer serves as a vital intracellular second messenger. A steady-state level of Cer is necessary for cell proliferation and differentiation (8,9), while excessive amounts of Cer are often toxic and can result in cellular apoptosis (10). High concentrations of Cer can result in the induction of cell death, leading to tissue damage and organ failure, such as what is observed in retinal degeneration Sph) is the major source for Sph 1-phosphate production, which has an opposing role to Cer and provides cytoprotection. Here, we investigated the role of Cer in human RPE-derived ARPE19 cells under hydrogen peroxide-induced oxidative stress, and show that Cer and hexosyl-Cer levels increase in the oxidatively stressed ARPE19 cells, which can be prevented by overexpression of lysosomal ASAH1. This study demonstrates that oxidative stress generates sphingolipid death mediators in retinal cells and that induction of ASAH1 could rescue retinal cells from oxidative stress by hydrolyzing excess Overexpression of acid ceramidase (ASAH1) protects retinal cells (ARPE19) from oxidative stress. J. Lipid Res. 2019. 60: 30-43. Abstract Over 11 million people in the United States alone have some form of age-related macular degeneration (AMD). Oxidative stress, cell death, and the degeneration of retinal pigment epithelial (RPE) cells contribute to AMD pathology. Recent evidence suggests that ceramide (Cer), a cellular sphingolipid mediator that acts as a second messenger to induce apoptosis, might play a role in RPE cell death. The lysosomal breakdown of Cer by acid ceramidase [N-acylsphingosine amidohydrolase (ASAH)1] into sphingosine (Supplementary key words N-acylsphingosine amidohydrolase 1 • ceramide • hexosyl-ceramide • lysosome, retinal pigment epithelium • retinal degeneration • age-related macular degeneration

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“…Surprisingly, very little is known about the role of SPLs in the human cornea and the mechanisms of corneal fibrosis. In fact, there are only two reports that showed the presence of Sphingosine kinase-1 ( SphK1), Sphingosine kinase-2 (SphK2), S1P 1-3,5 receptor proteins [ 11 ] and mRNA [ 39 ] in cultured human primary corneal fibroblasts. Expression of S1P receptor’s mRNA have also been noted by [ 40 ], in cultured corneal epithelial cells mimicking wound healing responses in vivo .…”

Section: Introductionmentioning

confidence: 99%

Unravelling the interplay of sphingolipids and TGF-β signaling in the human corneal stroma

et al. 2017

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PurposeTo delineate the role of Sphingolipids (SPLs) in the human cornea and their cross-talks with transforming growth factor beta (TGF-β) in order to develop novel, non-invasive therapies.MethodsHuman corneal fibroblasts (HCFs) were harvested from healthy donors, stimulated with Vitamin C to promote extracellular matrix assembly, treated with exogenous sphingosine-1-phosphate (S1P) or sphingosine kinase inhibitor 2 (SPHK I2) and isolated after 4 weeks for further analysis.ResultsData showed that S1P led to a significant decrease in cellular migration where SPHK I2 just delayed it for 24h. Significant modulation of the sphingolipid pathway was also noted. Sphingosine kinase-1 (SphK1) was significantly downregulated upon exogenous stimulation with S1P at a concentration of 5μM and Sphingosine kinase-2 (SphK2) was also significantly downregulated at concentrations of 0.01μM, 0.1μM, and 5μM; whereas no effects were observed upon stimulation with SPHK I2. S1PR3 was significantly downregulated by 0.1μM and 5μM S1P and upregulated by 5μM and 10μM SPHK I2. Furthermore, both S1P and SPHK I2 regulated corneal fibrosis markers such as alpha-smooth muscle actin, collagen I, III, and V. We also investigated the interplay between two TGF-β isoforms and S1P/SPHK I2 treatments and found that TGF-β1 and TGF-β3 were both significantly upregulated with the 0.1μM S1P but were significantly downregulated with the 5μM S1P concentration. When TGF-β1 was compared directly to TGF-β3 expression, we observed that TGF-β3 was significantly downregulated compared to TGF-β1 in the 5μM concentration of S1P. No changes were observed upon SPHK I2 treatment.ConclusionOur study delineates the role of sphingolipids in the human cornea and highlights their different activities based on the cell/tissue type.

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Analysis of sphingolipids in human corneal fibroblasts from normal and keratoconus patients

Cited by 20 publications

References 68 publications

Pisum sativum Defensin 1 Eradicates Mouse Metastatic Lung Nodules from B16F10 Melanoma Cells

Pisum sativum Defensin 1 Eradicates Mouse Metastatic Lung Nodules from B16F10 Melanoma Cells

Overexpression of acid ceramidase (ASAH1) protects retinal cells (ARPE19) from oxidative stress

Unravelling the interplay of sphingolipids and TGF-β signaling in the human corneal stroma

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