Search results for: Shuailin LI

Authors: Shanhe LIU, Shuailin LI, Shao-Lun HSU, Berthold Seitz, Shweta Suiwal, Tanja Stachon, Nóra Szentmáry

Abstract:

Purpose: Congenital aniridia is a rare ocular disorder with partial or complete absence of the iris in most cases and is frequently accompanied by aniridia-associated keratopathy (AAK). Evidence from prior studies suggests increased susceptibility of corneal limbal stromal cells to inflammatory stimuli, in which an increased apoptotic rate may play a significant role. This study aimed to investigate apoptosis in primary aniridia limbal stromal cells and to assess changes in p21 and p27 levels in response to lipopolysaccharide (LPS)-induced inflammation in vitro. Methods: Primary human corneal fibroblasts were isolated from the limbal region of both aniridia (AN-LSCs; n=8) and healthy (LSCs; n=8) donors. The cells were treated with 0 µg/ml, 2.5 µg/ml, 10 µg/ml and 17.5 µg/ml LPS for 24 hours. Apoptosis was assessed by flow cytometry in each group. The expression levels of apoptosis-related genes CDKN1A (p21) and CDKN1B (p27) were measured by qPCR. p21 and p27 protein levels were analyzed by flow cytometry. Results: Flow cytometry revealed a significantly higher apoptotic rate in AN-LSCs, than in LSCs (p<0.0001). CDKN1A mRNA level and p21 protein level were significantly higher in AN-LSCs than in LSCs (p=0.0232, p=0.0003). In AN-LSCs, 17.5 µg/ml LPS treatment significantly increased the apoptotic rate (p=0.0007) but had no effect on the apoptotic rate in LSCs (p>0.05). In LSCs, 10 and 17.5 µg/ml LPS treatment significantly increased CDKN1B mRNA levels (p=0.0028, p=0.0019) without changes in p27 protein levels (p>0.05). In AN-LFC, all LPS concentrations significantly increased CDKN1B mRNA levels (p≤0.0051) without changes at the protein level (p>0.05). Conclusions: There is an increased apoptotic rate in limbal stromal cells of congenital aniridia patients, which is accompanied by an increased p21 protein level. AN-LSCs are more sensible to LPS-induced inflammation than normal controls, and LPS treatment triggers CDKN1B mRNA levels both in AN-LSCs and LSCs Further studies should clarify the specific changes in the apoptotic cascade and identify potential therapeutic targets in limbal stromal cells of patients with congenital aniridia, aiming to prevent or delay the progression of AAK.

Keywords: limbal fibroblasts, aniridia associated keratopathy, lipopolysaccharide, apoptosis

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Authors: Shuailin Li, Tanja Stachon, Fabian N. Fries, Zhen Li, Shanhe Liu, Shao-Lun Hsu, Berthold Seitz, Swarnali Kundu, Maryam Amini, Shweta Suiwal, Nóra Szentmáry

Abstract:

Purpose: Aniridia associated keratopathy (AAK) is a progressive condition commonly observed in individuals with congenital aniridia, with PAX6 haploinsufficiency. AAK can lead to limbal stem cell deficiency and progressive ocular surface damage. The dysfunction of limbal epithelial and stromal cells (LECs and LSCs) potentially plays a key role in AAK pathogenesis. Travoprost, a prostaglandin analog, has been shown to influence cellular behavior in various cell types, but its effects on primary aniridia LECs and LSCs remain unclear. This study aims to evaluate the impact of travoprost on cell viability, proliferation, and migration in LECs, in the siRNA-based limbal epithelial cell model and in primary aniridia LSCs, in vitro. Methods: Primary human LECs were extracted from heathy donors, and siRNA treatment was used to mimic PAX6 haploinsufficiency in congenital aniridia. Primary human LSCs were extracted from heathy and aniridia donors (AN-LSCs). LECs, LSCs and AN-LSCs were treated with 0.039-40 μg/ml travoprost, for 20 minutes. The XTT and BrdU assays were used to evaluate the effect of travoprost on cell viability and proliferation (n=7). Cell migration assay was performed following siRNA-based PAX6 knockdown and subsequent 0.313 and 0.156 μg/ml travoprost treatment for 20 minutes (n=5). Results: LECs and LSCs viability decreased significantly from 0.156 μg/ml and ANLSCs viability decreased significantly from 0.078 μg/ml travoprost concentration (p=0.0279, p<0.0001, p=0.0002). In all cell types, there was a stepwise decrease in cell viability, as the travoprost concentration increased (p<0.0001). Travoprost treatment did not change cell proliferation in LSCs (p≥0.0892). In LECs, 20 and 40 μg/ml travoprost concentration, in AN-LSCs 40 μg/ml travoprost concentration exhibited reduced cell proliferation, compared to untreated controls (p=0.0291, p=0.0041, p=0.0011). PAX6-knockdown LECs exhibited lower migration rates at 6, 12, and 24 hours (p=0.0015, p=0.0471, p=0.0009) than control siRNA treated LECs, following travoprost treatment. In contrast, AN-LSCs demonstrated higher migration rates at the same 3 time points, than LSCs, after treatment (p=0.0225, p=0.0383, p=0.0155). In addition, among AN-LSCs, migration rate at of the 0.313 μg/ml travoprost treated group at 6 hours was significantly higher, than in the untreated control group. Conclusions: Our results demonstrate that travoprost may exert different effects on LECs, PAX6-knockdown LECs, LSCs, and AN-LSCs regarding cell viability, proliferation, and migration. AN-LSCs appear to exhibit greater sensitivity to travoprost treatment, than LSCs, therefore, topical antiglaucomatous treatment should be selected with caution for patients with congenital aniridia. Further in vivo measurements are necessary to evaluate the potential role of travoprost on AAK.

Keywords: congenital aniridia, travoprost, primary limbal epithelial cells, primary limbal stromal cells

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Authors: Shao-Lun Hsu, Tanja Stachon, Fabian N. Fries, Zhen Li, Shuailin Li, Shanhe Liu, Berthold Seitz, Swarnali Kundu, Maryam Amini, Shweta Suiwal, Nóra Szentmáry

Abstract:

Purpose: Congenital aniridia is characterized by PAX6 haploinsufficiency, and aniridia-associated keratopathy (AAK). In AAK, limbal stem cell deficiency and impaired wound healing are wiPurpose: Congenital aniridia is characterized by PAX6 haploinsufficiency and aniridia-associated keratopathy (AAK). In AAK, limbal stem cell deficiency and impaired wound healing are widely observed in patients, which might be associated with an imbalanced retinoic acid (RA) signaling pathway. In the previous studies, we demonstrated the relationship between PAX6 and the altered expression levels of key markers in the RA signaling pathway to retinol treatment. The present study evaluates the gene and protein expression levels in an in vitro small interfering RNA (siRNA) PAX6 knockdown aniridia limbal epithelial cell model following retinoic acid treatment. This study targets the direct effects of active RA products and their association with key regulators of the RA signaling pathway in siRNA PAX6 knockdown LECs. Methods: Primary human limbal epithelial cells (LECs) were knocked down by siRNA treatment to mimic PAX6 deletion in congenital aniridia (n=8). This was followed by 0µM, 1µM, and 5µM retinoic acid treatment applied in both siRNA PAX6 control and knockdown groups. After 48h incubation, paired box 6 (PAX6), alcohol dehydrogenase 7 (ADH7), aldehyde dehydrogenase 1 family member A1 (ALDH1A1), cytochrome P450 family 26 subfamilies A member 1 (CYP26A1), retinol-binding protein 1 (RBP1), cellular retinoic acid binding protein 2 (CRABP2), fatty acid binding protein 5 (FABP5), retinoid X receptor alpha (RXRA), retinoid X receptor beta (RXRB), retinoic acid receptor alpha (RARA), retinoic acid receptor beta (RARB), peroxisome proliferator-activated receptor gamma (PPARG), vascular endothelial growth factor A (VEGFA) mRNA levels have been determined using qPCR and protein levels by ELISA or western blot. Results: PAX6, ADH7, ALDH1A1, FABP5 mRNA levels and PAX6, ADH7, FABP5, PPARG2 protein levels were significantly lower in the PAX6 knockdown group, than in controls (p<0.001, p=0.018, p=0.015, p<0.001; p<0.001, p=0.003, p<0.001, p=0.007). PPARG mRNA level was significantly higher in the PAX6 knockdown group than in controls (p=0.012). CYP26A1 mRNA expression was upregulated using 1 µM and 5 µM RA treatment in both the PAX6 control (p<0.001; p<0.001) and the PAX6 knockdown group (p=0.001; p=0.002). CRABP2 mRNA expression in the PAX6 knockdown group (p=0.02) and protein expression in both groups were downregulated using to 5 µM RA concentration (p=0.003; p=0.02). RARA mRNA expression in the PAX6 knockdown group (p=0.023), RARB mRNA expression in both groups (p=0.006, p=0.001), and RXRA protein expression in controls (p=0.007), were downregulated using 5 µM RA concentration. VEGFA mRNA expression in PAX6 controls was upregulated using 5 µM RA (p=0.041). FABP5 to CRAP2 ratio was higher in PAX6 controls than in the PAX6 knockdown group (p<0.001). Additionally, the FABP5 to CRAP2 ratio was only upregulated in PAX6 controls using 5 µM RA concentration but not in the PAX6 knockdown group (p<0.001). Conclusions: These results reveal a less-responsive FABP5 to CRABP2 ratio in PAX6 knockdown LECs following increased RA concentration, as well as altered expression of key regulators in the RA signaling pathway. Further investigations into the regulatory processes are required to elucidate the role of RA signaling in the development of AAK.

Keywords: congenital aniridia, paired box 6 gene, aniridia-associated keratopathy, retinoic acid signaling pathway

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