Para-Hydroxycinnamic Acid Mitigates Senescence and Inflammaging in Human Skin Models.

Para-hydroxycinnamic acid (pHCA) is one of the most abundant naturally occurring hydroxycinnamic acids, a class of chemistries known for their antioxidant properties. In this study, we evaluated the impact of pHCA on different parameters of skin aging in in vitro skin models after H2O2 and UV exposure. These parameters include keratinocyte senescence and differentiation, inflammation, and energy metabolism, as well as the underlying molecular mechanisms. Here we demonstrate that pHCA prevents oxidative stress-induced premature senescence of human primary keratinocytes in both 2D and 3D skin models, while improving clonogenicity in 2D. As aging is linked to inflammation, referred to as inflammaging, we analyzed the release of IL-6, IL-8, and PGE2, known to be associated with senescence. All of them were downregulated by pHCA in both normal and oxidative stress conditions. Mechanistically, DNA damage induced by oxidative stress is prevented by pHCA, while pHCA also exerts a positive effect on the mitochondrial and glycolytic functions under stress. Altogether, these results highlight the protective effects of pHCA against inflammaging, and importantly, help to elucidate its potential mechanisms of action.

Effects of Different Capsulotomy and Fragmentation Energy Levels on the Generation of Oxidative Stress Following Femtosecond Laser-Assisted Cataract Surgery.

PURPOSE: This study was conducted to evaluate the effects of different capsulotomy and fragmentation energy levels on the production of oxidative free radicals following femtosecond laser-assisted cataract surgery (FLACS) with a low-energy platform. METHODS: The experimental study included 60 porcine eyes (12 groups). In each group, capsulotomies with 90% or 150% energy, and fragmentations with 90%, 100%, or 150% energy or 150% with high spot density, respectively, were performed. Control samples were obtained from non-lasered eyes at the beginning (five eyes) and end (five eyes) of the experiment. In the clinical study, 104 eyes were divided into 5 groups, and they received conventional phacoemulsification (20 eyes), FLACS with 90% capsulotomy and 100% fragmentation energy levels without NSAIDs (16 eyes), FLACS with 90% (26 eyes) or 150% (22 eyes) capsulotomy energy levels, respectively, with a 100% fragmentation energy level and NSAIDs, and FLACS with 90% capsulotomy and 150% fragmentation energy levels and NSAIDs (20 eyes). Aqueous samples were analyzed for their malondialdehyde (MDA) and superoxide dismutase (SOD) levels. RESULTS: In the experimental study, there were no significant differences in the MDA and SOD levels between the groups with different capsulotomy energy levels. An increase in the fragmentation energy from 100% to 150% led to significantly higher MDA levels in the groups with both 90% (p = 0.04) and 150% capsulotomy energy levels (p = 0.03), respectively. However, increased laser spot densities did not result in significant changes in MDA or SOD levels. In the clinical study, all four of the FLACS groups showed higher MDA levels than the conventional group. Similarly, the increase in the fragmentation energy from 100% to 150% resulted in significantly elevated levels of MDA and SOD, respectively. CONCLUSIONS: Although increasing the FSL capsulotomy energy level may not have increased free radicals, higher fragmentation energy levels increased the generation of aqueous free radicals. However, fragmentation with high spot density did not generate additional oxidative stress. Increased spot density did not generate additional oxidative stress, and this can be helpful for dense cataracts.

The Effects of High Energy Capsulotomy on Aqueous Cytokine Profiles and Pupil Size During Femtosecond Laser-Assisted Cataract Surgery.

PURPOSE: To assess whether aqueous cytokine profiles and pupil size are altered when high capsulotomy energy is used in eyes undergoing femtosecond laser-assisted cataract surgery (FLACS), and if preoperative use of a topical non-steroidal anti-inflammatory drug (NSAID) has an effect on this. METHODS: This prospective study recruited 83 eyes (63 patients) that were allocated to four treatment groups: conventional phacoemulsification (n = 20 eyes); FLACS with 90% capsulotomy energy without NSAID pretreatment (n = 20 eyes); FLACS with 90% capsulotomy energy with NSAID pre-treatment (n = 21 eyes); and FLACS with 150% capsulotomy energy with NSAID pretreatment (n = 22 eyes). Aqueous humor was collected before and after phacoemulsification to assess cytokine profiles. Pupil size was measured before and after laser capsulotomy. RESULTS: FLACS increased aqueous concentrations of pros-taglandin E2 (PGE2), interferon γ (IFN-γ), and interleukin 6 (IL-6) compared to conventional phacoemulsification. However, when increasing capsulotomy energy from 90% to 150% (with topical NSAID pretreatment), there was no significant increase in aqueous concentrations of PGE2 (37.7 ± 21.7 vs 33.6 ± 27.6 pg/mL, P = .99), IFN-γ (3.6 ± 1.1 vs 3.6 ± 0.8 pg/mL, P = .99), or IL-6 (7.1 ± 2.9 vs 6.3 ± 2.4 pg/mL, P = .99). For 90% and 150% capsulotomy energy, there was significant miosis following laser capsulotomy. Increased PGE2 concentration was significantly correlated with a reduction in pupil area (r = -0.58, P < .001) and pupil diameter (r = -0.57, P < .001). However, when a topical NSAID was given preoperatively, there was no difference in the degree of miosis between the 90% and 150% capsulotomy energy groups. CONCLUSIONS: Pretreatment with a topical NSAID prevented a rise in PGE2, IFN-γ, and IL-6 levels and excessive miosis when a higher capsulotomy energy was used. When a topical NSAID is used preoperatively, it is safe to use higher capsulotomy energy settings (with a low pulse energy femtosecond laser system) to achieve a satisfactory capsulotomy. [J Refract Surg. 2022;38(9):587-594.].

Randomized Controlled Trial Comparing 1-Year Outcomes of Low-Energy Femtosecond Laser-Assisted Cataract Surgery versus Conventional Phacoemulsification.

Purpose: To compare 1-year clinical outcomes, phacoemulsification energy, aqueous profiles, and patient-reported outcomes of low-energy femtosecond laser-assisted cataract surgery (FLACS) vs. conventional phacoemulsification. Methods: The study is a randomized controlled trial (RCT) with paired-eye design. Eighty-five patients were randomized to receive FLACS (Ziemer LDV Z8) in one eye and conventional phacoemulsification in the fellow eye. Clinical data including phacoemulsification energy parameters (cumulative dissipated energy, phacoemulsification power, and phacoemulsification time), uncorrected and corrected distance visual acuities (UCDVA and BCDVA), manifest refraction spherical equivalent (MRSE), central corneal thickness (CCT), endothelial cell count (ECC), anterior chamber flare, and post-operative complications were obtained for 1 year. Aqueous humor was collected for the analysis of prostaglandin (PGE)2, cytokines and chemokines concentrations. Patients' reported-outcomes on surgical experiences were evaluated using an in-house questionnaire. Results: Compared to conventional phacoemulsification, the low-energy assisted FLACS group had significantly less ECC reduction at 3 months (1.5 ± 0.3% vs. 7.0 ± 2.4%; P < 0.01) and 1 year (8.2 ± 2.8% vs. 11.2 ± 3.6%; P = 0.03). There were no significant differences in the phacoemulsification energy parameters, UCDVA, BCDVA, MRSE, CCT, occurrence of post-operative complications between the 2 groups throughout post-operative 1 year. Patients' subjective surgical experiences, including the surgical duration and perceived inconvenience, were comparable between the 2 groups. FLACS resulted in significantly higher aqueous PGE2 (P < 0.01), interleukin (IL)-6 (P = 0.03), IL-8 (P = 0.03), and interferon (IFN)-γ (P = 0.04) concentrations and greater anterior chamber flare at 1 day (P = 0.02). Conclusions: Our RCT presented 1-year longitudinal clinical and laboratory data. The long-term ECC result was more favorable in low-energy FLACS. The rest of the intraoperative and post-operative outcomes, as well as patient-reported outcomes, were comparable between these two procedures.

Cellular therapy of corneal epithelial defect by adipose mesenchymal stem cell-derived epithelial progenitors.

BACKGROUND: Persistent epithelial defects (PED), associated with limbal stem cell deficiency (LSCD), require ocular surface reconstruction with a stable corneal epithelium (CE). This study investigated CE reformation using human adipose mesenchymal stem cells (ADSC), which derived epithelial progenitors via mesenchymal-epithelial transition (MET). METHODS: STEMPRO human ADSC were cultured with specific inhibitors antagonizing glycogen synthase kinase-3 and transforming growth factor-ß signaling, followed by culture under a defined progenitor cell targeted-epithelial differentiation condition to generate epithelial-like cells (MET-Epi), which were characterized for cell viability, mesenchymal, and epithelial phenotypes using immunofluorescence and flow cytometry. Tissue-engineered (TE) MET-Epi cells on fibrin gel were transplanted to corneal surface of the rat LSCD model caused by alkali injury. Epithelial healing, corneal edema, and haze grading, CE formation were assessed by fluorescein staining, slit lamp bio-microscopy, anterior segment optical coherence tomography, and immunohistochemistry. RESULTS: CD73high/CD90high/CD105high/CD166high/CD14negative/CD31negative human ADSC underwent MET, giving viable epithelial-like progenitors expressing δNp63, CDH1 (E-cadherin), epidermal growth factor receptor, integrin-ß4, and cytokeratin (CK)-5, 9. Under defined epithelial differentiation culture, these progenitors generated MET-Epi cells expressing cell junction proteins ZO1 and occludin. When transplanted onto rat corneal surface with LSCD-induced PED, TE-MET-Epi achieved more efficient epithelial healing, suppressed corneal edema, and opacities, when compared to corneas without treatment or transplanted with TE-ADSC. CE markers (CK3, 12, and CDH1) were expressed on TE-MET-Epi-transplanted corneas but not in other control groups. CONCLUSION: Human ADSC-derived epithelial-like cells, via MET, recovered the CE from PED associated with LSCD. ADSC can be a viable adult stem cell source for potential autologous epithelial cell-based therapy for corneal surface disorders.

Changes in aqueous oxidative stress, prostaglandins, and cytokines: Comparisons of low-energy femtosecond laser-assisted cataract surgery versus conventional phacoemulsification.

PURPOSE: To compare the aqueous oxidative stress, prostaglandin E2 (PGE2), and cytokine levels after low-energy femtosecond laser-assisted cataract surgery versus conventional phacoemulsification, and to evaluate the effect of a nonsteroidal antiinflammatory drug (NSAID) on the aqueous profiles. SETTING: Singapore National Eye Center, Singapore. DESIGN: Randomized controlled trial. METHODS: Thirty-five patients were randomized to receive conventional phacoemulsification in one eye and femtosecond laser-assisted cataract surgery in the fellow eye. Another matched prospective cohort of 35 patients was included to receive femtosecond laser-assisted cataract surgery with a preoperative NSAID. Aqueous humor was collected after the laser was used or at the beginning of surgery (conventional phacoemulsification), and at the end of phacoemulsification. The levels of aqueous malondialdehyde (MDA), PGE2, and cytokines, chemokines, and growth factors were analyzed. The postoperative aqueous flare levels were evaluated. RESULTS: Compared with the conventional group, the femtosecond-assisted group had a significantly higher PGE2 (P = .01) and interleukin-1 receptor antagonist levels (P = 0.04). The preoperative NSAID significantly reduced the PGE2 surge (P = .002) and the mean reduction in pupil diameter (P = .02). The MDA concentrations before phacoemulsification were 0.24 µmol/L ± 0.18 (SD), 0.51 ± 0.41 µmol/L and 0.59 ± 0.52 µmol/L for the conventional, femtosecond-assisted, and femtosecond-assisted NSAID groups, respectively (P = .42). After phacoemulsification, the PGE2 and MDA levels increased in all groups. The MDA induction was significantly correlated with the phacoemulsification time (P = .002). The postoperative flare was insignificantly higher in the femtosecond-assisted group than the conventional group. CONCLUSIONS: Compared with conventional phacoemulsification, the femtosecond laser-assisted cataract surgery performed with the Femto LDV Z8 laser platform induced a significantly higher PGE2 level. The MDA and postoperative aqueous flare level were insignificantly higher. The preoperative NSAID reduced the PGE2 surge and occurrence of intraoperative miosis. The oxidative stress induced during phacoemulsification was strongly correlated with phacoemulsification time.

Postnatal periodontal ligament as a novel adult stem cell source for regenerative corneal cell therapy.

Corneal opacities are a leading cause of global blindness. They are conventionally treated by the transplantation of donor corneal tissue, which is, restricted by a worldwide donor material shortage and allograft rejection. Autologous adult stem cells with a potential to differentiate into corneal stromal keratocytes (CSKs) could offer a suitable choice of cells for regenerative cell therapy. Postnatal periodontal ligament (PDL) contains a population of adult stem cells, which has a similar embryological origin as CSK, that is cranial neural crest. We harvested PDL cells from young adult teeth extracted because of non-functional or orthodontic reason and differentiated them towards CSK phenotype using a two-step protocol with spheroid formation followed by growth factor and cytokine induction in a stromal environment (human amnion stroma and porcine corneal stroma). Our results showed that the PDL-differentiated CSK-like cells expressed CSK markers (CD34, ALDH3A1, keratocan, lumican, CHST6, B3GNT7 and Col8A2) and had minimal expression of genes related to fibrosis and other lineages (vasculogenesis, adipogenesis, myogenesis, epitheliogenesis, neurogenesis and hematogenesis). Introduction of PDL spheroids into the stroma of porcine corneas resulted in extensive migration of cells inside the host stroma after 14-day organ culture. Their quiescent nature and uniform cell distribution resembled to that of mature CSKs inside the native stroma. Our results demonstrated the potential translation of PDL cells for regenerative corneal cell therapy for corneal opacities.

Inhibiting glycogen synthase kinase-3 and transforming growth factor-ß signaling to promote epithelial transition of human adipose mesenchymal stem cells.

BACKGROUND: This study was aimed to investigate the epithelial differentiation of human adipose-derived mesenchymal stem cells (ADSCs) by inhibiting glycogen synthase kinase-3 (GSK3) and transforming growth factor ß (TGFß) signaling. METHODS AND RESULTS: STEMPRO human ADSCs at passage 2 were treated with CHIR99021 (GSK3 inhibitor), E-616452 (TGFß1 receptor kinase inhibitor), A-83-01 (TGFß type 1 receptor inhibitor), valproic acid (histone deacetylase inhibitor), tranylcypromine (monoamine oxidase inhibitor) and all-trans retinoic acid for 72 h. The mesenchymal-epithelial transition was shown by down-regulation of mesenchymal genes (Slug, Zinc Finger E-box Binding Homeobox 1 ZEB1, integrin α5 ITGA5 and vimentin VIM) and up-regulation of epithelial genes (E-cadherin, Epithelial Cell Adhesion Molecule EpCAM, Zonula Occludens-1 ZO-1, occludin, deltaN p63 δNp63, Transcription Factor 4 TCF4 and Twist Family bHLH Transcription Factor TWIST), compared to untreated ADSCs. Cell morphology and stress fiber pattern were examined and the treated cells became less migratory in scratch wound closure assay. The formation of cell junction complexes was observed under transmission electron microscopy. Global gene expression using GeneChip® Human Genome U133 Array (Affymetrix) showed that the treatment up-regulated 540 genes (containing genes for cell cycle, cytoskeleton reorganization, chemotaxis, epithelium development and regulation of cell migration) and down-regulated 483 genes. CONCLUSION: Human ADSCs were transited to epithelial lineage by inhibiting GSK3 and TGFß signaling. It can be an adult stem cell source for epithelial cell-based therapy.

Decellularization of human stromal refractive lenticules for corneal tissue engineering.

Small incision lenticule extraction (SMILE) becomes a procedure to correct myopia. The extracted lenticule can be used for other clinical scenarios. To prepare for allogeneic implantation, lenticule decellularization with preserved optical property, stromal architecture and chemistry would be necessary. We evaluated different methods to decellularize thin human corneal stromal lenticules created by femtosecond laser. Treatment with 0.1% sodium dodecylsulfate (SDS) followed by extensive washes was the most efficient protocol to remove cellular and nuclear materials. Empty cell space was found inside the stroma, which displayed aligned collagen fibril architecture similar to native stroma. The SDS-based method was superior to other treatments with hyperosmotic 1.5 M sodium chloride, 0.1% Triton X-100 and nucleases (from 2 to 10 U/ml DNase and RNase) in preserving extracellular matrix content (collagens, glycoproteins and glycosaminoglycans). The stromal transparency and light transmittance was indifferent to untreated lenticules. In vitro recellularization showed that the SDS-treated lenticules supported corneal stromal fibroblast growth. In vivo re-implantation into a rabbit stromal pocket further revealed the safety and biocompatibility of SDS-decellularized lenticules without short- and long-term rejection risk. Our results concluded that femtosecond laser-derived human stromal lenticules decellularized by 0.1% SDS could generate a transplantable bioscaffold with native-like stromal architecture and chemistry.

Regulation of Toll-like receptor expression in human conjunctival epithelial cells.

Previous studies showed marked decrease of multiple Toll-like receptor (TLR) expression in corneal and conjunctival epithelial cells upon culture in vitro. The aim of this study was to identify factor(s) which regulate TLR expression. Primary human conjunctival epithelial cells and immortal conjunctival (IOBA-NHC) and corneal epithelial cell lines (HCET) were used. The effect of various cytokines, hypoxia, mechanical wounding, and airlifting culture on TLR expression was examined by quantitative PCR and western blot analysis. Ligand stimulated TLR activation was analyzed. TLR mRNA expression increased modestly when cultured monolayered cells were stimulated by TNF-α, IL-1α, IL-1ß, IL-6, IL-8, IFN-γ (about 2-fold), hypoxia (2.1- to 4.8-fold selectively), and wounding (3.1- to 9.3-fold). In airlifted multilayered cells, TLR expression increased 7.8- to 25.9-fold compared to monolayered cells. Airlifted cells showed increased response to low concentrations of lipopolysaccharide (LPS) and peptidoglycan (PGN) stimulation. NF κ B inhibition prevented the formation of cell sheets and led to the collapse of already-formed multilayered structure and the simultaneous reduction of TLR mRNA level. In conclusion, our study showed that the conjunctival epithelial cell expressed TLR was sensitive to various stimulants, and a multilayered epithelium-like structure was needed to maintain TLR expression.

S100A expression in normal corneal-limbal epithelial cells and ocular surface squamous cell carcinoma tissue.

PURPOSE: To study the expression and cellular distribution of multiple S100A genes and proteins in normal corneal-limbal epithelium and ocular surface squamous cell carcinoma (SCC) tissue. METHODS: Normal corneal-limbal tissue was obtained from the Lions Eye Bank, Tampa, FL. Ocular surface SCC tissues were excised from patients undergoing surgery at Singapore National Eye Centre. S100A mRNA expression was measured by quantitative PCR. S100 protein distribution was determined by immunofluorescent staining analysis. RESULTS: Twelve S100 mRNAs were identified in human corneal and limbal epithelial cells. S100A2, A6, A8, A9, A10, and A11 mRNA was expressed at high level, while S100A1, A3, A4, A5, A6, A7, and A12 mRNA expression was low. The intracellular localization of S100A2, A6, A8, A9, A10 and A11 protein was determined in normal corneal-limbal and SCC tissues. S100A2 and S100A10 proteins were enriched in basal limbal epithelial cells of the normal tissue. S100A8 and S100A9 were found only at the surface of peripheral corneal and limbal epithelium. S100A6 was uniformly found at the plasma membrane of corneal and limbal epithelial cells. S100A11 was found at the supralayer limbal epithelial cells adjacent to the conjunctiva. SCC tissue showed typical pathological changes with expression of cytokeartin (CK) 14 and CK4 in the epithelial cells. All SCC epithelial cells were positive of S100A2, S100A10, S100A6 and S100A11 staining. Intracellular staining of S100A8 and S100A9 was found in several layers of SCC epithelium. Expression of S100A2 and S100A10 decreased dramatically in cultured limbal epithelial cells with increased passaging, which was accompanied by a small increase of S100A9 mRNA, with no changes of S100A8 gene expression. Serum and growth hormone depletion of the culture serum caused a small reduction of S100A2 and S100A10 gene expression, which was accompanied by a small increase of S100A9 mRNA while no changes of S100A8 expression was measured. CONCLUSIONS: Normal corneal and limbal epithelial cells express a broad spectrum of S100 genes and proteins. Ocular surface SCC express high levels of S100A2, S100A10, S100A8 and S100A9 proteins. The expression of S100A2 and S100A10 is associated with limbal epithelial cell proliferation and differentiation.