A bioengineering approach to Schlemm's canal-like stem cell differentiation for in vitro glaucoma drug screening.

Human Schlemm's canal (HSC) cells are critical for understanding outflow physiology and glaucoma etiology. However, primary donor cells frequently used in research are difficult to isolate. HSC cells exhibit both vascular and lymphatic markers. Human adipose-derived stem cells (ADSCs) represent a potential source of HSC due to their capacity to differentiate into both vascular and lymphatic endothelial cells, via VEGF-A and VEGF-C. Shear stress plays a critical role in maintaining HSC integrity, function, and PROX1 expression. Additionally, the human trabecular meshwork (HTM) microenvironment could provide cues for HSC-like differentiation. We hypothesize that subjecting ADSCs to VEGF-A or VEGF-C, shear stress, and co-culture with HTM cells could provide biological, mechanical, and cellular cues necessary for HSC-like differentiation. To test this hypothesis, effects of VEGF-A, VEGF-C, and shear stress on ADSC differentiation were examined and compared to primary HSC cells in terms of cell morphology, and HSC marker expression using qPCR, immunoblotting, and immunocytochemistry analysis. Furthermore, the effect of co-culture with HTM cells on porous scaffolds on ADSC differentiation was studied. Treatment with VEGF-C under shear stress is effective in differentiating ADSCs into PROX1-expressing HSC-like cells. Co-culture with HTM cells on porous scaffolds leads to HTM/ADSC-derived HSC-like constructs that regulate through-flow and respond as expected to dexamethasone. STATEMENT OF SIGNIFICANCE: We successfully generated human Schlemm's canal (HSC) like cells from adipocyte-derived stem cells induced by biochemical and biomechanical cues as well as bioengineered human trabecular meshwork (HTM) on micropatterned, porous SU8 scaffolds. These stem cell-derived HSC-like cells co-cultured with HTM cells on SU8 scaffolds can regulate through-flow, and in particular, are responsive to steroid treatment as expected. These findings show that ADSC-derived HSC-like cells have the potential to recreate the ocular outflow pathway for in vitro glaucoma drug screening. To the best of our knowledge, it is the very first time to demonstrate derivation of Schlemm's canal-like cells from stem cells. It provides an important alternative source to primary Schlemm's canal cells that are very difficult to be isolated and cultured from human donors.

Regulatory Roles of Anoctamin-6 in Human Trabecular Meshwork Cells.

Purpose: Trabecular meshwork (TM) cell volume is a determinant of aqueous humor outflow resistance, and thereby IOP. Regulation of TM cell volume depends on chloride ion (Cl-) release through swelling-activated channels (ICl,Swell), whose pore is formed by LRRC8 proteins. Chloride ion release through swelling-activated channels has been reported to be regulated by calcium-activated anoctamins, but this finding is controversial. Particularly uncertain has been the effect of anoctamin Ano6, reported as a Ca2+-activated Cl- (CaCC) or cation channel in other cells. The current study tested whether anoctamin activity modifies volume regulation of primary TM cell cultures and cell lines. Methods: Gene expression was studied with quantitative PCR, supplemented by reverse-transcriptase PCR and Western immunoblots. Currents were measured by ruptured whole-cell patch clamping and volume by electronic cell sizing. Results: Primary TM cell cultures and the TM5 and GTM3 cell lines expressed Ano6 3 to 4 orders of magnitude higher than the other anoctamin CaCCs (Ano1 and Ano2). Ionomycin increased cell Ca2+ and activated macroscopic currents conforming to CaCCs in other cells, but displayed significantly more positive mean reversal potentials (+5 to +12 mV) than those displayed by ICl,Swell (-14 to -21 mV) in the same cells. Nonselective CaCC inhibitors (tannic acid>CaCCinh-A01) and transient Ano6 knockdown strongly inhibited ionomycin-activated currents, ICl,Swell and the regulatory volume response to hyposmotic swelling. Conclusions: Ionomycin activates CaCCs associated with net cation movement in TM cells. These currents, ICl,Swell, and cell volume are regulated by Ano6. The findings suggest a novel clinically-relevant approach for altering cell volume, and thereby outflow resistance, by targeting Ano6.

Ascorbic acid modulation of iron homeostasis and lysosomal function in trabecular meshwork cells.

PURPOSE: To investigate the antioxidant properties and biological functions of ascorbic acid (AA) on trabecular meshwork (TM) cells. METHODS: Primary cultures of porcine TM cells were supplemented for 10 days with increasing concentrations of AA. Antioxidant properties against cytotoxic effect of H2O2 were evaluated by monitoring cell viability. Redox-active iron was quantified using calcein-AM. Intracellular reactive oxygen species (iROS) production was quantified using H2DCFDA. Ferritin and cathepsin protein levels were analyzed by Western blot. Autophagy was evaluated by monitoring lipidation of LC3-I to LC3-II. Lysosomal proteolysis and cathepsins activities were quantified using specific fluorogenic substrates. RESULTS: AA exerts a dual effect against oxidative stress in TM cells, acting as an anti-oxidant or a pro-oxidant, depending on the concentration used. The pro-oxidant effect of AA was mediated by free intracellular iron and correlated with increased protein levels of ferritin and elevated iROS. In contrast, antioxidant properties correlated with lower ferritin and basal iROS content. Ascorbic acid supplementation also caused induction of autophagy, as well as increased lysosomal proteolysis, with the latter resulting from higher proteolytic activation of lysosomal cathepsins in treated cultures. CONCLUSIONS: Our results suggest that the reported decrease of AA levels in plasma and aqueous humor can compromise lysosomal degradation in the outflow pathway cells with aging and contribute to the pathogenesis of glaucoma. Restoration of physiological levels of vitamin C inside the cells might improve their ability to degrade proteins within the lysosomal compartment and recover tissue function.

Pharmacological regulation of SPARC by lovastatin in human trabecular meshwork cells.

PURPOSE: Statins have been shown to increase aqueous outflow facility. The matricellular protein SPARC (secreted protein acidic and rich in cysteine) is a critical mediator of aqueous outflow and intraocular pressure (IOP). Here, we examine the effects of lovastatin on SPARC expression in trabecular meshwork (TM) cells, exploring the molecular mechanisms involved. METHODS: Primary cultured human TM cells were incubated for 24, 48, and 72 hours with 10 µM lovastatin. In separate cultures, media was supplemented with either farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP) for the duration of the 72-hour time point experiment. Trabecular meshwork cells were also pretreated for 24 hours with lovastatin followed by 24-hour stimulation with 3 ng/mL TGF-ß2. Cell lysates and media were harvested and relative mRNA and protein level changes were determined. Krüppel-like factor 4 (KLF4) localization in normal human anterior segments was examined by immunofluorescence. Adenovirus expressing human KLF4 was used and relative changes in SPARC mRNA and protein levels were assessed. RESULTS: Incubating TM cells with lovastatin suppressed SPARC mRNA and protein levels. This effect was reversed upon media supplementation with GGPP but not FPP. Pretreating cells with lovastatin inhibited TGF-ß2 induction of SPARC. The KLF4 transcription factor was expressed throughout the TM and the inner and outer walls of Schlemm's canal. Lovastatin treatment upregulated KLF4 mRNA and protein levels. Overexpression of KLF4 downregulated SPARC expression. CONCLUSIONS: Collectively, our data identify lovastatin as an important pharmacological suppressor of SPARC expression in TM cells, and provide further insight into the molecular mechanisms mediating statin enhancement of aqueous outflow facility.

Preventive effects of omega-3 and omega-6 Fatty acids on peroxide mediated oxidative stress responses in primary human trabecular meshwork cells.

Pathologic processes in glaucoma include increased apoptosis, accumulation of extracellular material in the trabecular meshwork and optic nerve, condensations of the cytoskeleton and precocious cellular senescence. Oxidative stress was shown to generate these alterations in primary ocular cells. Fatty acids omega-3 and -6 are alleged to constitute a prophylaxis against these deleterious effects. Here, we tested actual preventive effects omega-3 and -6 against peroxide induced stress responses in primary human trabecular meshwork cells. Changes of mitochondrial activity, proliferation, heat shock proteins, extracellular matrix components, and inflammatory markers were evaluated. Alterations of the cytoskeleton were evaluated by phalloidin labeling. Here we report a repressive effect of omega-6 on metabolic activity and proliferation, which was not detected for omega-3. Both agents were able to prevent the anti-proliferative effect of H2O2, but only omega-3 prevented metabolic repression. Expression of heat shock protein 27 was unaltered by both fatty acids, whereas heat shock protein 90 was significantly induced by both. Omega-6 increased fibronectin and connective tissue growth factor synthesis, as well as the amount of secreted fibronectin. Omega-3, instead, induced plasminogen activator inhibitor 1 synthesis. H2O2 further increased fibronectin production in omega-6 supplemented cells, which was not the case in omega-3 treated cells. H2O2 stimulation of plasminogen activator inhibitor 1 and connective tissue growth factor was repressed by both fatty acids. Both fatty acids appeared to abolish H2O2 mediated stimulation of nuclear factor κB and IL-6, but not IL-1α and IL-8. H2O2 induced formation of cross-linked actin networks and stress fibers, which was reduced by preemptive application of omega-3. Omega-6, in contrast, had no protective effect on that, and even seemed to promote condensation. Based on the observed side effects of omega-6, omega-3 appears to be the more beneficial fatty acid in respect of prophylactic intake for prevention of a glaucomatous disease.

Intracameral voriconazole: in vitro safety for human ocular cells.

Fungal keratitis is a sight-threatening infection of the cornea. It sometimes leads to loss of the eye. Despite an expanding range of fungal pathogens, there are only few therapeutic agents for its treatment available. Voriconazole is a second-generation synthetic triazole with a broad action against yeasts and molds. The current study investigates the safety of voriconazole for intracameral application in a cell culture model. Endothelial toxicity of voriconazole was evaluated in cultured human corneas. Possible toxic effects of voriconazole (10 microg /mL-10mg/mL) in corneal endothelial cells (CEC), primary human trabecular meshwork cells (TMC), and primary human retinal pigment epithelium (RPE) cells were evaluated after 24h and under conditions of inflammatory stress by treatment with tumor-necrosis-factor alpha (TNF-alpha), lipopolysaccharides (LPS), or interleukin-6 (IL-6) and hydrogen peroxide. Toxicity was evaluated by tetrazolium dye-reduction assay, and cell viability was quantified by a microscopic live-dead assay. No corneal endothelial toxicity could be detected after 30 days of treatment with 250 microg /mL of voriconazole. Concentrations up to 1mg/mL had no influence on CEC, TMC, or RPE cell proliferation, or on cell viability when administered for 24h. Hydrogen peroxide exposure did not increase cellular toxicity of voriconazole at concentrations from 10 to 250 microg /mL. After preincubation with TNF-alpha, LPS, or IL-6 for 24h and subsequent voriconazole treatment for 24h, no significant decrease in proliferation or viability was observed. This study showed no significant toxicity for voriconazole on CEC, TMC, RPE cells, or human corneal endothelium when administered in therapeutic concentrations up to 250 microg /mL.

Electron probe X-ray microanalysis of intact pathway for human aqueous humor outflow.

Intraocular pressure (IOP) is regulated by the resistance to outflow of the eye's aqueous humor. Elevated resistance raises IOP and can cause glaucoma. Despite the importance of outflow resistance, its site and regulation are unclear. The small size, complex geometry, and relative inaccessibility of the outflow pathway have limited study to whole animal, whole eye, or anterior-segment preparations, or isolated cells. We now report measuring elemental contents of the heterogeneous cell types within the intact human trabecular outflow pathway using electron-probe X-ray microanalysis. Baseline contents of Na(+), K(+), Cl(-), and P and volume (monitored as Na+K contents) were comparable to those of epithelial cells previously studied. Elemental contents and volume were altered by ouabain to block Na(+)-K(+)-activated ATPase and by hypotonicity to trigger a regulatory volume decrease (RVD). Previous results with isolated trabecular meshwork (TM) cells had disagreed whether TM cells express an RVD. In the intact tissue, we found that all cells, including TM cells, displayed a regulatory solute release consistent with an RVD. Selective agonists of A(1) and A(2) adenosine receptors (ARs), which exert opposite effects on IOP, produced similar effects on juxtacanalicular (JCT) cells, previously inaccessible to functional study, but not on Schlemm's canal cells that adjoin the JCT. The results obtained with hypotonicity and AR agonists indicate the potential of this approach to dissect physiological mechanisms in an area that is extremely difficult to study functionally and demonstrate the utility of electron microprobe analysis in studying the cellular physiology of the human trabecular outflow pathway in situ.

Alterations in human trabecular meshwork cell homeostasis by selenium.

Epidemiological evidence indicates that selenium supplementation may increase risk for glaucoma and ocular hypertension. The purpose of this study was to determine the effects of selenium on trabecular meshwork cells, a likely site of pathology for glaucoma. Human trabecular meshwork (HTM) cells and human umbilical vein endothelial cells (HUVECs) were treated with selenium (MSeA) at or near physiologically relevant concentrations. Selenium uptake by cells was monitored using mass spectrometry. Alterations in protein secretion, intracellular signaling, and cell morphology were monitored; and the role of integrin signaling in MSeA-induced morphological alterations was investigated using divalent cation treatments. Radiolabeling was used to assess protein synthesis and secretion, while luciferase and MTT assays monitored total cellular ATP and cell viability, respectively. Whereas detectible changes in intracellular selenium were observed after exposure to 1-10 microM MSeA for 24hr, the majority remained in the conditioned medium. Selenium-induced morphological changes (< or =3 hr) occurred before alterations in protein secretion and intracellular signaling (3-6 hr). Zinc treatment prevented selenium-mediated alterations in protein secretion and changes in cell-matrix adhesion. MSeA treatment (5 microM) led to a 60% decrease in protein synthesis after 3 hr and a 30% reduction in secretion, although significant alterations in cell viability and total ATP were not observed after MSeA treatment. Selenium altered several indicators of HTM cell homeostasis, but did not affect viability at physiologically relevant doses. Similar results with HUVECs have implications for understanding selenium's mechanisms of action as an anti-angiogenic agent.

Primary trabecular meshwork cells incubated in human aqueous humor differ from cells incubated in serum supplements.

PURPOSE: To determine whether aqueous humor, the in vivo source of nutrients for trabecular meshwork cells, alters cellular and molecular characteristics in primary trabecular monolayer cell cultures when compared with standard culture conditions. METHODS: Human primary trabecular meshwork cell cultures were grown in DMEM supplemented with 50% human aqueous humor (DMEM-AH), heat-denatured DMEM-AH, 10% fetal bovine serum (DMEM-FBS, the standard culture supplement), or heat-denatured DMEM-FBS. Confluent trabecular cells were assayed for cell propagation and morphology for 21 days. Protein expression profiles of trabecular cell lysates were analyzed by two-dimensional polyacrylamide gel electrophoresis. Western blot analysis was used to determine the protein expression of myocilin and TIMP-1 in conditioned media collected from trabecular cells at 5, 10, 15, and 21 days. Myocilin expression was also analyzed by Western immunoblots after addition of dexamethasone (10(-7) M) or ascorbic acid (29 mg/dL). RESULTS: Trabecular cells supplemented with DMEM-AH for 21 days showed decreased cell proliferation when compared with DMEM-FBS (11% vs. 141%). Cellular morphology was also altered: Trabecular cells incubated in DMEM-AH showed larger-, broader-, and flatter-appearing cells than did the more spindle-shaped cells grown in DMEM-FBS. Protein profiles of trabecular cell lysates isolated from cells incubated in DMEM-AH differed from those incubated in DMEM-FBS. In DMEM-AH-conditioned medium, myocilin expression was increased and TIMP-1 expression was decreased at day 21. Induction of myocilin by dexamethasone was observed in conditioned medium isolated from cells treated with DMEM-FBS (442%), but only a 10% increase in myocilin was observed beyond the normal induction in DMEM-AH. Daily administration of ascorbic acid to DMEM-AH failed to increase myocilin expression beyond that obtained with DMEM-AH. CONCLUSIONS: Addition of human aqueous humor rather than FBS to trabecular monolayer cell cultures triggers significant changes in cellular and molecular characteristics. The protein component of aqueous humor is responsible for these changes. Aqueous humor supplementation may maintain cultured trabecular cells in a more physiologic state.

Selenium's effects on MMP-2 and TIMP-1 secretion by human trabecular meshwork cells.

PURPOSE: Because of the observed increase in incidence of glaucoma among some individuals taking selenium as a dietary supplement, the present study was undertaken to investigate mechanisms of selenium-induced changes in homeostasis of human trabecular meshwork (HTM) cells. Specifically, the impact of selenium on matrix metalloproteinases (MMPs), their inhibitors (tissue inhibitors of metalloproteinases; TIMPs), and the second messengers that regulate MMP expression was investigated in an HTM cell culture model. METHODS: HTM cell cultures were treated with an organic selenium compound (methyl seleninic acid), and changes in secretion and activity of MMPs and TIMPs were analyzed by Western blot and zymography. Changes in extracellular-signal-related kinases 1 and 2 (ERK1/2) and phospho-ERK1/2 levels were monitored by Western blot analysis of whole-cell lysates prepared from selenium-treated cells. Photographs of cultures over time were used to document selenium-induced changes in cell morphology. RESULTS: Treatment of HTM cells with selenium for 24 hours at doses ranging from 1 to 10 micro M caused a dose-dependent decrease in the secretion of MMP-2 and TIMP-1. Treatment for 6 hours revealed a significant decrease in MMP-2 and TIMP-1 at the highest dose. MMP-1, -3, and -9 and TIMP-2 were either not detected or their secretion was not consistently influenced by selenium treatment. Selenium treatment caused a significant decrease in ERK1/2 phosphorylation, but no change in overall ERK protein levels. Selenium treatment resulted in dose-dependent, reversible changes in HTM cell-matrix associations. CONCLUSIONS: Selenium-induced changes in MMP-2/TIMP-1 secretion may alter the balance of extracellular matrix turnover in the conventional outflow pathway and cause an increase in intraocular pressure that eventually leads to glaucoma.

Ocular hypotensive FP prostaglandin (PG) analogs: PG receptor subtype binding affinities and selectivities, and agonist potencies at FP and other PG receptors in cultured cells.

Natural prostaglandins (PGs) such as PGD2, PGE2, PGF2(2alpha), and PGI2 exhibited the highest affinity for their respective cognate receptors, but were the least selective agents when tested in receptor binding assays. Travoprost acid ([+]-fluprostenol) was the most FP-receptor-selective compound, exhibiting a high affinity (Ki = 35 +/- 5 nM) for the FP receptor, and minimal affinity for DP (Ki = 52,000 nM), EP1 (Ki = 9540 nM), EP3 (Ki = 3501 nM), EP4 (Ki = 41,000 nM), IP (Ki > 90,000 nM), and TP (Ki = 121,000 nM) receptors. Travoprost acid was the most potent PG analog tested in FP receptor functional phosphoinositide turnover assays in the following cell types: human ciliary muscle (EC50 = 1.4 nM), human trabecular meshwork (EC50 = 3.6 nM), and mouse fibroblasts and rat aortic smooth muscle cells (EC50 = 2.6 nM). Although latanoprost acid exhibited a relatively high affinity for the FP receptor (Ki = 98 nM), it had significant functional activity at FP (EC50 = 32-124 nM) and EP1 (EC50 = 119 nM) receptors. Bimatoprost acid was less selective, exhibiting a relatively high affinity for the FP (Ki = 83 nM), EP1 (Ki = 95 nM), and EP3 (Ki = 387 nM) receptors. Bimatoprost acid exhibited functional activity at the EP1 (EC50 = 2.7 nM) and FP (EC50 = 2.8-3.8 nM in most cells) receptors. Bimatoprost (nonhydrolyzed amide) also behaved as an FP agonist at the cloned human FP receptor (EC50 = 681 nM), in h-TM (EC50 = 3245 nM) and other cell types. Unoprostone and S-1033 bound with low affinity (Ki = 5.9 microM to > 22 microM) to the FP receptor, were not selective, but activated the FP receptor. In conclusion, travoprost acid has the highest affinity, the highest FP-receptor-selectivity, and the highest potency at the FP receptor as compared to the other ocular hypotensive PG analogs known so far, including free acids of latanoprost, bimatoprost, and unoprostone isopropyl ester.

Human trabecular meshwork cells as a thyroid hormone target tissue: presence of functional thyroid hormone receptors.

PURPOSE: To determine whether human trabecular meshwork cells (HTM) are a potential target tissue for thyroid hormone (3,3',5-triiodothyronine, T3). METHODS: Cultured HTM were assayed for the presence of thyroid hormone receptors (TRs) and retinoid X receptors (RXRs) by reverse-transcriptase polymerase chain reaction (RT-PCR) to detected TR and RXR mRNA, and by immunohistochemistry to detect nuclear TR and RXR proteins. Functionality of the TR was determined by analysis of 125I-T3 binding affinity and capacity in HTM nuclear extracts. Effects of T3 on modulation of hyaluronic acid (HA) levels in HTM were measured as a function of dose and duration of T3 administration. RESULTS: Analysis of RT-PCR and immunohistochemistry demonstrated that cultured HTM expressed TRalpha1, TRalpha2, and TRbeta1 but not TRbeta2; and RXRalpha but not RXRbeta and RXRgamma isoforms. Saturation binding and analysis of 125I-T3 to HTM nuclear extracts revealed Kd of 57 pM. The number of T3 binding sites extrapolated from a Scatchard plot was 7.3 x 10(10)/microg of HTM nuclear protein extract. T3 supplementation reduced the concentration of HA in the cell medium by 32-43% compared to cells grown in the absence of T3. CONCLUSIONS: Cultured HTM express three TR isoforms and one RXR isoform, bind T3 with an affinity similar to that of TR in responsive cells, and modulate their HA production in response to T3. These findings indicate that the human trabecular meshwork tissue has the capacity to respond to thyroid hormones.