L-carnitine suppresses transient receptor potential vanilloid type 1 activity and myofibroblast transdifferentiation in human corneal keratocytes.

Corneal stromal wound healing is a well-balanced process promoted by overlapping phases including keratocyte proliferation, inflammatory-related events, and tissue remodeling. L-carnitine as a natural antioxidant has shown potential to reduce stromal fibrosis, yet the underlying pathway is still unknown. Since transient receptor potential vanilloid 1 (TRPV1) is a potential drug target for improving the outcome of inflammatory/fibrogenic wound healing, we investigated if L-carnitine can mediate inhibition of the fibrotic response through suppression of TRPV1 activation in human corneal keratocytes (HCK). We determined TRPV1-induced intracellular calcium transients using fluorescence calcium imaging, channel currents by planar patch-clamping, and cell migration by scratch assay for wound healing. The potential L-carnitine effect on TRPV1-induced myofibroblast transdifferentiation was evaluated by immunocytochemical detection of alpha smooth muscle actin. RT-PCR analysis confirmed TRPV1 mRNA expression in HCK. L-carnitine (1 mmol/l) inhibited either capsaicin (CAP) (10 µmol/l), hypertonic stress (450 mOsmol/l), or thermal increase (>43 °C) induced Ca2+ transients and corresponding increases in TRPV1-induced inward and outward whole-cell currents. This was accompanied by suppression of injury-induced increases in myofibroblast transdifferentiation and cell migration. In conclusion, L-carnitine contributes to inhibit stromal scarring through suppressing an injury-induced intrinsic TRPV1 activity that is linked with induction of myofibroblast transdifferentiation in HCK cells.

Ascorbate-induced oxidative stress mediates TRP channel activation and cytotoxicity in human etoposide-sensitive and -resistant retinoblastoma cells.

There are indications that pharmacological doses of ascorbate (Asc) used as an adjuvant improve the chemotherapeutic management of cancer. This favorable outcome stems from its cytotoxic effects due to prooxidative mechanisms. Since regulation of intracellular Ca2+ levels contributes to the maintenance of cell viability, we hypothesized that one of the effects of Asc includes disrupting regulation of intracellular Ca2+ homeostasis. Accordingly, we determined if Asc induced intracellular Ca2+ influx through activation of pertussis sensitive Gi/o-coupled GPCR which in turn activated transient receptor potential (TRP) channels in both etoposide-resistant and -sensitive retinoblastoma (WERI-Rb1) tumor cells. Ca2+ imaging, whole-cell patch-clamping, and quantitative real-time PCR (qRT-PCR) were performed in parallel with measurements of RB cell survival using Trypan Blue cell dye exclusion. TRPM7 gene expression levels were similar in both cell lines whereas TRPV1, TRPM2, TRPA1, TRPC5, TRPV4, and TRPM8 gene expression levels were downregulated in the etoposide-resistant WERI-Rb1 cells. In the presence of extracellular Ca2+, 1 mM Asc induced larger intracellular Ca2+ transients in the etoposide-resistant WERI-Rb1 than in their etoposide-sensitive counterpart. With either 100 µM CPZ, 500 µM La3+, 10 mM NAC, or 100 µM 2-APB, these Ca2+ transients were markedly diminished. These inhibitors also had corresponding inhibitory effects on Asc-induced rises in whole-cell currents. Pertussis toxin (PTX) preincubation blocked rises in Ca2+ influx. Microscopic analyses showed that after 4 days of exposure to 1 mM Asc cell viability fell by nearly 100% in both RB cell lines. Taken together, one of the effects underlying oxidative mediated Asc-induced WERI-Rb1 cytotoxicity stems from its promotion of Gi/o coupled GPCR mediated increases in intracellular Ca2+ influx through TRP channels. Therefore, designing drugs targeting TRP channel modulation may be a viable approach to increase the efficacy of chemotherapeutic treatment of RB. Furthermore, Asc may be indicated as a possible supportive agent in anti-cancer therapies.

Onion Epithelial Membrane Scaffolds Transfer Corneal Epithelial Layers in Reconstruction Surgery.

Plants and their extracts have been used especially in China for more than ten centuries for preventing and treating disease. However, there are only few reports describing their use in animal cell culture and tissue transplantation. In this study, onion epithelial membranes (OEM) is used as scaffolds to support cultures of a variety of cells such as fibroblasts and epithelial cells notably; they maintain the phenotypic characteristics of corneal epithelial cells. This improvement includes preservation of the proliferative potential and stemness of rabbit corneal epithelial cells (RCECs). Such an outcome suggests that this cost-effective technology warrants further evaluation to determine if OEM is a viable candidate for use as scaffolds in corneal epithelial transplantation surgery. To test this possibility, rabbit corneal epithelial cells expanded on OEM are transplanted to treat corneal epithelial defects in limbal stem cell deficient rabbits. This procedure is successful because it shortens the time required for wound healing to restore losses in corneal epithelial integrity, and forms a more compact and stratified epithelium framework than the untreated group. Ultimately, should they be proven to be effective in other relevant animal model systems, their usefulness for treating wounds in a clinical setting warrants consideration.

Gastro-protective effects of isobrucein B, a quassinoid isolated from Picrolemma sprucei.

Infusions of Picrolemma sprucei roots, stems and leaves are used in traditional medicine throughout the Amazon region from the Guianas to Brazil and Peru in the treatment of gastritis, intestinal helminths and malaria. As there are no studies describing its mode of action in providing a gastroprotective effect, we determined herein that one of the main constituents found in P. sprucei infusions, the quassinoid isobrucein B (IsoB), reduces some of the pathophysiological effects in a mouse model of non-steroidal anti-inflammatory drug (NSAID)-induced gastritis and provides mechanisms of action. Then, IsoB (1.17 g) was isolated from the roots and stems (6.5 kg) of P. sprucei. Its structure was confirmed by 1D and 2D (1)H and (13)C NMR, ESI-tof-MS, IR and UV. C57BL/6 strain mice were subcutaneously injected with IsoB (0.5-5 mg kg(-1)) or vehicle before oral administration of indomethacin and sacrificed later at different time points. Gastric damage was assessed by measuring lesion length. Leukocyte migration was evaluated based on leukocyte rolling and adhesion using intravital microscopy in the mesenteric microcirculation and tissue MPO activity. Stomach extract cytokine (TNFα, IL-1ß and KC/CXCL1) and prostaglandin E2 (PGE2) levels were measured by ELISA and RIA, respectively. IsoB pre-treatment (0.5-5.0 mg kg(-1)) significantly reduced the formation of indomethacin-induced stomach lesions in a dose-dependent manner. The decrease in stomach lesions was associated with less observed leukocyte rolling, decreased leukocyte adhesion and less neutrophil infiltration (MPO activity). IsoB (1 mg kg(-1)) pre-treatment did not prevent indomethacin-induced decreases in stomach PGE2 levels. However, IL-1ß and KC/CXCL1 levels were inhibited by this same IsoB dosage, whereas TNF-α was unchanged. IsoB may be a prototypic compound to provide protective effects against NSAID-induced gastritis and possibly other gastropathies. Moreover, IsoB gastroprotective action may be due to a reduction in IL-1ß and KC/CXCL1 production/release and leukocyte rolling, adhesion and migration.

TRPV1 activation is required for hypertonicity-stimulated inflammatory cytokine release in human corneal epithelial cells.

PURPOSE: To determine whether hypertonic stress promotes increases in inflammatory cytokine release through transient receptor potential vanilloid channel type 1 (TRPV1) signaling pathway activation in human corneal epithelial cells (HCECs). METHODS: Hyperosmotic medium was prepared by supplementing isotonic Ringers solution with sucrose. Ca2+ signaling was measured in fura2-AM-loaded HCECs using a single-cell fluorescence imaging system. Western blot analysis evaluated the phosphorylation status of EGFR, ERK, p38 MAPK, and nuclear factor (NF)-κB. ELISA assessed the effect of TRPV1 activation on the release of IL-6 and IL-8. RESULTS: A 450 mOsm hypertonic stress elicited 2-fold Ca2+ transients that were suppressed by the TRPV1-selective antagonists capsazepine and JYL 1421. Such transients were enhanced by PGE2. Hypertonicity-induced EGF receptor (EGFR) transactivation was suppressed by preincubating HCECs with capsazepine, matrix metalloproteinase 1 (MMP1) inhibitor TIMP-1, broad-spectrum MMP inhibitor GM 6001, heparin-bound (HB)-EGF inhibitor CRM 197, or EGFR inhibitor AG 1478. ERK and p38 MAPK and NF-κB activation after EGFR transactivation occurred in tonicity and in a time-dependent manner. Hypertonicity-induced increases in IL-6 and IL-8 releases were suppressed by exposure to capsazepine, AG 1478, ERK inhibitor PD 98059, p38 inhibitor SB 203580, or NF-κB inhibitor PDTC. CONCLUSIONS: Hypertonic stress-elicited TRPV1 channel stimulation mediates increases in a proinflammatory cytokine IL-6 and a chemoattractant IL-8 by eliciting EGFR transactivation, MAPK, and NF-κB activation. Selective drug modulation of either TRPV1 activity or its signaling mediators may yield a novel approach to suppressing inflammatory responses occurring in dry eye syndrome.

Emodin suppression of ocular surface inflammatory reaction.

PURPOSE: To determine whether a Chinese herbal medicine component, emodin, suppresses inflammatory/fibrogenic reaction in cultured subconjunctival fibroblasts and reduces injury-induced increases in ocular surface inflammation in mice. METHODS: Effects of emodin were measured in human subconjunctival fibroblasts on proliferation and migration with colorimetry and scratch wound assay, respectively. Neovascularization was evaluated using an endothelial cell-fibroblast coculture model. Proinflammatory mediator and extracellular matrix component gene and protein expression was characterized with real-time reverse transcription-polymerase chain reaction, enzyme immunoassay, and immunocytochemistry, respectively. Western blotting and immunohistochemistry evaluated the activation of nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK). In a mouse corneal alkali-burn model, the effects of emodin on ocular surface inflammation and fibrosis were evaluated. RESULTS: Emodin suppressed tumor necrosis factor alpha (TNF-alpha)-induced fibroblast migration and fibronectin deposition in vitro. VEGF induced neovascularization but did not affect cell proliferation and collagen type 1 production. Monocyte/macrophage-chemoattractant protein-1 gene and protein expression declined. Emodin inhibited TNF-alpha-induced NF-kappaB p65 and JNK activation but did not affect transforming growth factor beta1-induced Smad2/3 signaling. In vivo, emodin inhibited proinflammatory and fibrogenic reactions. CONCLUSIONS: Emodin suppressed in vitro TNF-alpha-induced stimulation of proinflammatory reaction. In a mouse ocular alkali burn model, this herbal component lessened inflammation and scarring. Additional studies are warranted to evaluate the therapeutic potential of emodin in lessening ocular tissue inflammation and resultant fibrosis after injury.

Differential dependence of regulatory volume decrease behavior in rabbit corneal epithelial cells on MAPK superfamily activation.

We characterized the dependence of hypotonicity-induced regulatory volume decrease (RVD) responses on mitogen-activated protein kinase (MAPK) pathway signaling in SV40-immortalized rabbit corneal epithelial cells (RCEC). Following calcein-AM loading, RVD was monitored using a microplate fluorescence reader. Western blot analysis determined MAPK activation. After 30 min, the RVD response restored the relative cell volume to nearly isotonic values, whereas it was inhibited when cells were bathed either in a Cl- -free solution or with the Cl- -channel inhibitors: 5-nitro-2-(3-phenylpropylamino)benzoic acid or niflumic acid. Similar declines occurred with either a high-K+ (20 mM) supplemented solution or the K+ channel inhibitor 4-aminopyridine. Activation of extracellular signal-regulated kinase (ERK), p38, and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) was time and tonicity-dependent. Stimulation of ERK and SAPK/JNK was maximized earlier than that of p38. Activation of ERK and SAPK/JNK was insensitive to Cl- and K+ channel inhibitors, whereas inhibition with either PD98059 or SP600125, respectively, blocked RVD. However, inhibition of p38 with SB203580had no effect on RVD. Suppression of RVD instead blocked p38 activation. Differences in the dependence of RVD activation on Erk1/2 and p38 signaling were validated in dominant negative (d/n)-Erk1 and d/n-p38 cells. Volume-sensitive Cl- and K+ channel activation contributes, in concert, to RVD in RCEC. Therefore, swelling-induced ERK and SAPK/JNK stimulation precedes Cl- and K+ channel activation, whereas p38 activation occurs as a consequence of RVD.

Genipin suppresses subconjunctival fibroblast migration, proliferation and myofibroblast transdifferentiation.

PURPOSE: Inchin-ko-to is a herbal medicine which has therapeutic effects in ameliorating liver fibrosis or cholestatic liver diseases. Its main bioactive component is genipin, which is an intestinal bacterial metabolite of this medication. Accordingly, we determined whether or not Inchin-ko-to suppresses in a wound healing model subconjunctival fibroblast (SCF) migration proliferation and myofibroblast transdifferentiation since an inhibitory effect could be of value in improving trabeculotomy outcome. METHODS: Effects of genipin on SCF cell migration were examined subsequent to wounding confluent monolayer cultures. Alamar blue staining evaluated the effects of genipin (0-50 microg/ml) on fibroblast cell proliferation. Immunostaining determined alpha-smooth muscle actin (alphaSMA) expression. Western blotting evaluated (alphaSMA) expression and phospho-Smad2 formation. Real-time RT-PCR evaluated TGFbeta1 and collagen Ialpha2 mRNA expression. Enzyme-immunoassay determined culture medium collagen I content. RESULTS: Genipin suppressed wound-induced cell migration and proliferation. It also decreased collagen type I TGFbeta1 and alphaSMA mRNA and protein expression. Smad2 signaling was inhibited by genipin in a dose-dependent manner. CONCLUSION: Genipin suppresses injury-induced fibrogenic responses in SCFs. This result suggests that the herbal medicine Inchin-ko-to might have therapeutic value following trabeculotomy.

Genipin suppression of fibrogenic behaviors of the alpha-TN4 lens epithelial cell line.

PURPOSE: To determine in a lens epithelial cell line, alpha-TN4, whether genipin, an intestinal metabolite component of the herbal medicine inchin-ko-to, suppresses profibrogenic myofibroblast generation and upregulation of fibrogenic cytokines and to evaluate the potential benefit of the medicine in preventing posterior capsule opacification (PCO). SETTING: Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. METHODS: In this study, alpha-TN4 cell proliferation, migration, and expression of alpha-smooth muscle actin (alpha-SMA), the hallmark of myofibroblast generation, were assayed with a colorimetric assay, scratch wound assay, immunohistochemistry, and Western blot analysis. Gene expression of transforming growth factor-beta1 (TGF-beta1) and connective tissue growth factor (CTGF) was characterized with real-time reverse transcription-polymerase chain reaction. In addition, p38 mitogen-activated protein kinase (p 38 MAPK), extracellular signal-regulated kinase (ERK) limb, and Smad signalings were evaluated by Western blotting and immunohistochemistry. Cytotoxicity of genipin was evaluated using a commercial colorimetric assay kit for nuclear matrix protein 41/7 (NMP41/7) in culture medium. RESULTS: Genipin suppressed cell proliferation and migration in association with inhibition of Smad and p38 MAPK phosphorylation, although ERK signaling was enhanced. Genipin suppressed mRNA expression of TGF-beta1 and CTGF. Cytoplasmic fiber formation declined based on less intense alpha-SMA immunocytochemical staining. However, alpha-SMA protein expression was actually not altered. This negative result suggests that genipin attenuated formation of alpha-SMA-containing cytoskeleton. Treatment of the cells with genipin for 48 hours did not increase the release of NMP41/7 to the medium, indicating this compound is not cytotoxic. CONCLUSION: Because genipin suppressed alpha-TN4 lens cell fibrogenic behaviors, it may be of therapeutic value in preventing PCO.

EGF suppresses hydrogen peroxide induced Ca2+ influx by inhibiting L-type channel activity in cultured human corneal endothelial cells.

Endogenous generated hydrogen peroxide during eye bank storage limits viability. We determined in cultured human corneal endothelial cells (HCEC) whether: (1) this oxidant induces elevations in intracellular calcium concentration [Ca2+]i; (2) epidermal growth factor (EGF) medium supplementation has a protective effect against peroxide mediated rises in [Ca2+]i. Whereas pathophysiological concentrations of H2O2 (10 mM) induced irreversible large increases in [Ca2+]i, lower concentrations (up to 1 mM) had smaller effects, which were further reduced by exposure to either 5 microM nifedipine or EGF (10 ng ml(-1)). EGF had a larger protective effect against H2O2-induced rises in [Ca2+]i than nifedipine. In addition, icilin, the agonist for the temperature sensitive transient receptor potential protein, TRPM8, had complex dose-dependent effects (i.e. 10 and 50 microM) on [Ca2+]i. At 10 microM, it reversibly elevated [Ca2+]i whereas at 50 microM an opposite effect occurred suggesting complex effects of temperature on endothelial viability. Taken together, H2O2 induces rises in [Ca2+]i that occur through increases in Ca2+ permeation along plasma membrane pathways that include L-type Ca2+ channels as well as other EGF-sensitive pathways. As EGF overcomes H2O2-induced rises in [Ca2+]i, its presence during eye bank storage could improve the outcome of corneal transplant surgery.

Osmosensitive taurine transporter expression and activity in human corneal epithelial cells.

PURPOSE: To characterize in SV40-immortalized human corneal epithelial cells (tHCEC) osmosensitive taurine transporter gene and protein expression as well as its functional activity. To evaluate whether medium supplementation with taurine improves cell viability during a hypertonic challenge. METHODS: tHCEC were preincubated for up to 48 hours in hypertonic DMEM medium (i.e., up to 500 mosmol/kg). Taurine uptake was monitored through measurements of intracellular [3H]taurine accumulation. Gene and protein expression was detected by Northern and Western blot analyses, respectively. An amino acid analyzer measured intracellular cold taurine content. The live/dead assay evaluated with confocal microscopy determined cell viability. RESULTS: Na+-dependent taurine uptake occurred in an isotonic (310 mosmol/kg) medium. The apparent Michaelis-Menten constant, K(t), for taurine was 4.6 micro M, and uptake increased as a function of exposure time and rises in osmolality. Exposure for 12 hours to a 450 mosmol/kg medium increased uptake by 4.1-fold. However, after 48 hours of exposure to this medium, taurine uptake returned to its isotonic level. With time, biphasic changes occurred in taurine transporter gene and protein expression and taurine transport activity as well as elevating intracellular taurine content by 4.5-fold. Taurine medium supplementation for 48 hours improved cell viability. CONCLUSIONS: tHCEC express Na+-dependent osmosensitive taurine transport activity. The hypertonic-induced biphasic effects on gene and protein expression as well as transport activity suggest feedback regulation of these responses. Rises in intracellular taurine do not appear to be essential for osmoregulation, but instead enhance cell survival perhaps through a membrane stabilizer or an antioxidant effect.