Development of a novel in vivo corneal fibrosis model in the dog.

The aim of this study was to develop a novel in vivo corneal model of fibrosis in dogs utilizing alkali burn and determine the ability of suberanilohydroxamic acid (SAHA) to inhibit corneal fibrosis using this large animal model. To accomplish this, we used seven research Beagle dogs. An axial corneal alkali burn in dogs was created using 1 N NaOH topically. Six dogs were randomly and equally assigned into 2 groups: A) vehicle (DMSO, 2 µL/mL); B) anti-fibrotic treatment (50 µM SAHA). The degree of corneal opacity, ocular health, and anti-fibrotic effects of SAHA were determined utilizing the Fantes grading scale, modified McDonald-Shadduck (mMS) scoring system, optical coherence tomography (OCT), corneal histopathology, immunohistochemistry (IHC), and transmission electron microscopy (TEM). The used alkali burn dose to produce corneal fibrosis was well tolerated as no significant difference in mMS scores between control and treatment groups (p = 0.89) were detected. The corneas of alkali burned dogs showed significantly greater levels of α-smooth muscle actin, the fibrotic marker, than the controls (p = 0.018). Total corneal thickness of all dogs post-burn was significantly greater than baseline OCT images irrespective of treatment (p = 0.004); TEM showed that alkali burned corneas had significantly greater minimum and maximum interfibrillar distances than the controls (p = 0.026, p = 0.018). The tested topical corneal alkali burn dose generated significant opacity and fibrosis in dog corneas without damaging the limbus as evidenced by histopathology, IHC, TEM, and OCT findings, and represents a viable large animal corneal fibrosis in vivo model. Additional in vivo SAHA dosing studies with larger sample size are warranted.

Decorin biology, expression, function and therapy in the cornea.

Decorin is a small leucine-rich proteoglycan (SLRP) that plays a vital role in many important cellular processes in several tissues including the cornea. A normal constituent of the corneal stroma, decorin is also found in the majority of connective tissues and is related structurally to other small proteoglycans. It interacts with various growth factors such as epidermal growth factor (EGF) and transforming growth factor beta (TGFß) to regulate processes like collagen fibrillogenesis, extracellular matrix (ECM) compilation, and cell-cycle progression. Studies have linked decorin dysregulation to delayed tissue healing in patients with various diseases including cancer. In the cornea, decorin is involved in the regulation of transparency, a key function for normal vision. It has been reported that mutations in the decorin gene are associated with congenital stromal dystrophy, a disease that leads to corneal opacity and visual abnormalities. Decorin also antagonizes TGFß in the cornea, a central regulatory cytokine in corneal wound healing. Following corneal injury, increased TGFß levels induce keratocyte transdifferentiation to myofibroblasts and, subsequently, fibrosis (scarring) in the cornea. We recently reported that decorin overexpression in corneal fibroblasts blocks TGFß-driven myofibroblast transformation and fibrosis development in the cornea in vitro suggesting that decorin gene therapy can be used for the treatment of corneal scarring in vivo.

Role of transforming growth factor Beta in corneal function, biology and pathology.

Transforming growth factor-beta (TGFbeta) is a pleiotropic multifunctional cytokine that regulates several essential cellular processes in many parts of the body including the cornea. Three isoforms of TGFbeta are known in mammals and the human cornea expresses all of them. TGFbeta1 has been shown to play a central role in scar formation in adult corneas whereas TGFbeta2 and TGFbeta3 have been implicated to play a critical role in corneal development and scarless wound healing during embryogenesis. The biological effects of TGFbeta in the cornea have been shown to follow Smad dependent as well as Smad-independent signaling pathways depending upon cellular responses and microenvironment. Corneal TGFbeta expression is necessary for maintaining corneal integrity and corneal wound healing. On the other hand, TGFbeta is perhaps the most important cytokine in the pathogenesis of fibrotic disease in the cornea. Although the transformation of keratocytes to myofibroblasts induced by TGFbeta is largely believed to cause corneal fibrosis or scarring, the precise molecular mechanism(s) involved in this process is still unknown. Currently no drugs are available to treat corneal scarring effectively without causing significant side effects. Many approaches to treat TGFbeta-mediated corneal scarring are under investigation. These include blocking of TGFbeta, TGFbeta receptor, TGFbeta function and/or TGFbeta maturation. Other strategies such as modulating keratocyte proliferation, apoptosis, transcription and DNA condensation are also being investigated. The potential of gene therapy to neutralize the pathologic effects of TGFbeta has also been demonstrated recently.

Sleep-wakefulness in alcohol preferring and non-preferring rats following binge alcohol administration.

The alcohol-preferring (P) rat is a valid animal model of alcoholism. However, the effect of alcohol on sleep in P or alcohol non-preferring (NP) rats is unknown. Since alcohol consumption has tremendous impact on sleep, the present study compared the effects of binge alcohol administration on sleep-wakefulness in P and NP rats. Using standard surgical procedures, the P and NP rats were bilaterally implanted with sleep recording electrodes. Following post-operative recovery and habituation, pre-ethanol (baseline) sleep-wakefulness was electrographically recorded for 48 h. Subsequently, ethanol was administered beginning with a priming dose of 5 g/Kg followed by two doses of 2 g/Kg every 8 h on the first day and three doses of 3 g/Kg/8 h on the second day. On the following day (post-ethanol), undisturbed sleep-wakefulness was electrographically recorded for 24 h. Our initial results suggest that, during baseline conditions, the time spent in each of the three behavioral states: wakefulness, non-rapid eye movement (NREM) sleep and REM sleep, was comparable between P and NP rats. However, the P rats were more susceptible to changes in sleep-wakefulness following 2 days of binge ethanol treatment. As compared to NP rats, the P rats displayed insomnia like symptoms including a significant reduction in the amount of time spent in NREM sleep coupled with a significant increase in wakefulness on post-ethanol day. Subsequent analysis revealed that binge ethanol induced increased wakefulness and reduced NREM sleep in P rats occurred mainly in the dark period. This is the first study that: (1) demonstrates spontaneous sleep-wake profile in P and NP rats, and (2) compares the effects of binge ethanol treatment on sleep in P and NP rats. Our results suggest that, as compared to NP rats, the P rats were more susceptible to sleep disruptions after binge ethanol treatment. In addition, the P rats exhibited insomnia-like symptoms observed during abstinence from alcohol in human subjects.

Proinflammatory chemokine induction in keratocytes and inflammatory cell infiltration into the cornea.

PURPOSE: To determine the effect of interleukin (IL)-1alpha and tumor necrosis factor (TNF)-alpha on cytokine, chemokine, and receptor expression in corneal stromal cells; the effect of corneal scrape injury on monocyte chemotactic and activating factor (MCAF) expression and monocyte-macrophage influx into the stroma; and the effect of MCAF and granulocyte colony-stimulating factor (G-CSF) microinjection on inflammatory cell infiltration into the stroma. METHODS: Gene array technology was used to evaluate changes in cytokine, chemokine, and receptor gene expression in stromal fibroblasts in response to IL-1alpha and TNFalpha. Expression of MCAF mRNA and protein was monitored with an RNase protection assay and Western blot analysis, respectively. Keratocyte MCAF protein expression in the rabbit cornea was detected with immunocytochemistry. After epithelial scrape injury, monocytes-macrophages were detected in rabbit corneas, by immunocytochemistry for monocyte-macrophage antigen. Inflammatory cell infiltration after MCAF and G-CSF microinjection into the stroma of mouse corneas was monitored with hematoxylin and eosin staining. RESULTS: IL-1alpha or TNFalpha upregulated the expression of several proinflammatory chemokines in stromal fibroblasts in culture. These included G-CSF, MCAF, neutrophil-activating peptide (ENA-78), and monocyte-derived neutrophil chemotactic factor (MDNCF). MCAF mRNA upregulation was confirmed by RNase protection assay, and MCAF protein was detected by Western blot analysis. MCAF protein was detected in keratocytes at 4 hours and 24 hours after epithelial injury, but not in keratocytes in the unwounded cornea. Corneal epithelial injury triggered the influx of monocytes-macrophages into the corneal stroma in the rabbit. Microinjection of MCAF and G-CSF into mouse cornea resulted in the influx of monocytes-macrophages and granulocytes, respectively, into the stroma. CONCLUSIONS: Proinflammatory chemokine induction in keratocytes is mediated by IL-1alpha and TNFalpha. The proinflammatory chemokines produced by the keratocytes probably trigger the influx of inflammatory cells into the stroma after epithelial injury associated with corneal surgery, contact lenses, or trauma.

The corneal wound healing response: cytokine-mediated interaction of the epithelium, stroma, and inflammatory cells.

The corneal wound healing cascade is complex and involves stromal-epithelial and stromal-epithelial-immune interactions mediated by cytokines. Interleukin-1 appears to be a master modulator of many of the events involved in this cascade. Keratocyte apoptosis is the earliest stromal event noted following epithelial injury and remains a likely target for modulation of the overall wound healing response. Other processes such as epithelial mitosis and migration, stromal cell necrosis, keratocyte proliferation, myofibroblast generation, collagen deposition, and inflammatory cell infiltration contribute to the wound healing cascade and are also likely modulated by cytokines derived from corneal cells, the lacrimal gland, and possibly immune cells. Many questions remain regarding the origin and fate of different cell types that contribute to stromal wound healing. Over a period of months to years the cornea returns to a state similar to that found in the unwounded normal cornea.

The wound healing response after laser in situ keratomileusis and photorefractive keratectomy: elusive control of biological variability and effect on custom laser vision correction.

Biological diversity in the wound healing response is thought to be a major factor limiting the predictability of the outcome of refractive surgical procedures such as laser in situ keratomileusis and photorefractive keratectomy. Corneal wound healing is critical to the success of topography-linked or wave front-linked excimer laser ablation to optimize visual performance. This is because of the importance of retaining subtle features of custom ablation and the tendency of epithelial hyperplasia and stromal remodeling to obscure these features following either procedure. The corneal wound healing response is exceedingly complex. Keratocyte apoptosis, which occurs in response to epithelial injury, is the earliest observable event in the wound healing cascades and is therefore an excellent target for pharmacological intervention. Alterations of surgical technique can be designed to limit keratocyte apoptosis and the subsequent events in corneal wound healing. Abnormalities of the cascades could contribute to the pathogenesis of corneal diseases. For example, recent data have suggested that perturbation of the keratocyte apoptosis/mitosis balance could underlie the development of keratoconus in a proportion of patients.

Discoidin domain receptor (DDR) 1 and 2: collagen-activated tyrosine kinase receptors in the cornea.

Discoidin domain receptor (DDR) 1 and 2 have recently been found to serve as receptors for several collagen types. These receptors have been found to modulate cell proliferation and metalloprotease expression in response to collagen stimulation. The purpose of this study was to examine expression of DDR1 and DDR2 in the cornea and to determine the effect of several collagen types on proliferation and response to pro-apoptotic cytokines by corneal fibroblasts. DDR1 and DDR2 mRNAs were detected by RT-PCR. Proteins were detected by immunocytochemistry and immunoprecipitation with Western blotting. Cell proliferation in response to acetic acid-solubilized collagen type I, II, IV, IX or X was determined by cell counting. The effect of these collagen types on Fas-stimulating antibody-induced cell death was determined by trypan blue assay. DDR1 and DDR2 mRNAs were detected in each major human cell type of the cornea. Both were also detected in ex vivo human corneal epithelium. DDR1 and DDR2 proteins were detected in all three major cell types in culture and in human corneal tissue. Collagen types I, II, IV, IX and X stimulated proliferation, but had no effect on Fas-mediated apoptosis, of corneal fibroblasts. DDR1 and DDR2 tyrosine kinase receptors are expressed in the cornea. Collagen-stimulated mitosis of corneal fibroblasts in culture is likely mediated by the DDR receptors. Collagen had no effect on Fas-mediated apoptosis of corneal fibroblasts.

Caspase inhibitor z-VAD-FMK inhibits keratocyte apoptosis, but promotes keratocyte necrosis, after corneal epithelial scrape.

The purpose of this study was to determine whether the caspase inhibitor z-VAD-FMK could be applied topically prior to epithelial scrape injury to inhibit keratocyte apoptosis. Rabbit corneas were treated with z-VAD-FMK or vehicle alone prior to epithelial scrape injury. Cell fate was analysed at 4 hr after epithelial scrape using quantitative TUNEL assay, propidium iodide staining, and transmission electron microscopy. Less stained anterior stromal keratocytes were detected with the quantitative TUNEL assay in corneas pre-treated with z-VAD-FMK than in corneas pretreated with vehicle at 4 hr after epithelial scrape. This difference appeared to be confirmed by propidium iodide staining of keratocyte nuclei. It was observed that fewer nuclei were stained with propidium iodide in the DMSO vehicle treated corneas compared to the z-VAD-FMK treated corneas. Analysis of corneas with transmission electron microscopy, however, indicated that many anterior stromal keratocytes in corneas pretreated with z-VAD-FMK, but not vehicle, had cell morphologic changes more consistent with necrosis. Although pretreatment of corneas with the caspase inhibitor z-VAD-FMK inhibited keratocyte apoptosis detected with the TUNEL assay, transmission electron microscopy revealed that many anterior stromal keratocytes in z-VAD-FMK-treated corneas instead died by necrosis. Thus, z-VAD-FMK is unlikely to be useful to modulate corneal would healing through inhibition of keratocyte apoptosis induced by epithelial injury. The TUNEL assay should not be used to monitor cell fate without confirmation using analyses that also detect necrosis.

Defective keratocyte apoptosis in response to epithelial injury in stat 1 null mice.

Defects in apoptosis have been noted in signal transducer and activator of transcription (Stat) 1-null cells in vitro. The purpose of this study was to analyse the keratocyte apoptosis response that occurs in vivo in response to corneal epithelial injury in Stat 1null compared with control mice and to determine whether Stat 1null corneal fibroblasts have a defective response to death receptor activation in vitro. Corneal epithelial scrape injuries were performed in Stat 1-null and wild-type mice. Keratocyte apoptosis was monitored with the quantitative TUNEL assay and confirmed using transmission electron microscopy. Corneal fibroblast apoptosis in response to tumor necrosis factor (TNF) alpha, with and without inhibitors of nuclear factor kappa B (NF-kappaB) activation, was monitored using DNA laddering and the methylene blue assay. Significantly less keratocyte apoptosis was noted in Stat 1-null mice compared with wild-type controls. TNF alpha-induced apoptosis only occurred in wild-type mice in the presence of inhibitors of NF-kappaB activation. Corneal fibroblast TNF alpha-induced apoptosis was defective in Stat 1null corneal fibroblasts whether NF-kappaB activation was blocked or not. Stat 1 has an important role in the keratocyte apoptosis that occurs in response to corneal epithelial injury. Previous studies suggest that the defect is due to a lack of constitutive expression of caspases. This study demonstrates that this defect in apoptosis in Stat 1-null mice is present in vivo in Stat 1-null mice and suggests that Stat 1 could be a therapeutic target for transient inhibition of keratocyte apoptosis to modulate corneal wound healing.

Modulation of TNF-alpha-induced apoptosis in corneal fibroblasts by transcription factor NF-kappaB.

PURPOSE: Previous studies have suggested no role for tumor necrosis factor (TNF)-alpha in the modulation of apoptosis in corneal fibroblasts. However, recent investigations have demonstrated that nuclear factor (NF)-kappaB activation by TNF-alpha mediates negative apoptotic effects that must be blocked to unmask the apoptotic effects of TNF-alpha in vitro. The purpose of this study was to investigate the role of transcription factor NF-kappaB in the suppression of TNF-alpha-induced apoptosis of corneal fibroblasts. METHODS: mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) and RNase protection assay. Proteins were detected by immunocytochemistry and immunoprecipitation with Western blot analysis. Cell death was evaluated by trypan blue exclusion assay in corneal fibroblasts treated with TNF-alpha in presence or absence of the specific inhibitor of NF-kappaB activation, SN50, actinomycin D, or actinomycin D with dexamethasone, ketorolac tromethamine, or diclofenac sodium. Apoptosis was monitored by trypan blue exclusion, colorimetric cell assay, CPP32 activation assay, DNA fragmentation assay, and transmission electron microscopy. NF-KB activation was monitored using electrophoretic gel shift assay. RESULTS: TNF-alpha, TNF receptor (R)I, and TNFRII mRNAs were detected in all three cultured corneal cell types and in ex vivo corneal epithelium using RT-PCR. TNF-alpha mRNA was also detected in ex vivo corneal epithelium, corneal epithelial cells, and stromal fibroblasts with the RNase protection assay. TNF-alpha, TNFRI, and TNFRII proteins were detected by immunocytochemistry in all three major corneal cell types in human corneal tissue. TNF-alpha protein was also detected in ex vivo corneal epithelium, primary corneal epithelial cells, and primary stromal fibroblasts using immunoprecipitation and Western blot analysis. TNF-alpha stimulated corneal fibroblast cell death when NF-kappaB activation was blocked with actinomycin D or SN50. Enhanced cell death was noted with dexamethasone, ketorolac tromethamine, or diclofenac sodium when used in the presence, but not in the absence, of actinomycin D. A gel shift assay revealed induction of NF-KB by TNF-alpha and suppression of induction in the presence of actinomycin D or SN50, but not by the control peptide SN50M. CONCLUSIONS: The TNF-alpha receptor system is expressed in the cornea, and NF-kappaB activation is an important regulator of TNF-alpha-mediated corneal fibroblast apoptosis. Nonsteroidal anti-inflammatory agents or corticosteroids may potentiate corneal fibroblast apoptosis in response to cytokine stimulation.

Effect of PDGF, IL-1alpha, and BMP2/4 on corneal fibroblast chemotaxis: expression of the platelet-derived growth factor system in the cornea.

PURPOSE: The purpose of this study was to examine expression of platelet-derived growth factor (PDGF) and PDGF receptors in the human cornea and to study the effects of the PDGF isotypes on proliferation and chemotaxis of human corneal fibroblasts. The effects of interleukin (IL)-1alpha, bone morphogenic protein (BMP)2, and BMP4 on chemotaxis of human corneal fibroblasts were also studied. METHODS: mRNA expression was monitored with reverse transcription-polymerase chain reaction (RT-PCR) in primary cultured cells. Protein expression in fresh-frozen human corneal sections was studied with immunocytology. Chemotaxis was measured using a modified Boyden chamber, and proliferation was quantitated by cell counting. RESULTS: PDGF A, PDGF B, PDGF receptor alpha, and PDGF receptor beta mRNAs were detected in corneal epithelial cells, fibroblasts, and endothelial cells in culture. The proteins were expressed in each major cell type in human corneal sections, with PDGF A and PDGF B detected at high levels in the epithelial basement membrane. PDGF, BMP2, and BMP4 had attractive chemotactic effects on corneal fibroblasts, with the PDGF BB dimer having a significantly greater positive chemotactic effect than the other PDGF isotypes. Interleukin-1alpha had a repulsive chemotactic effect on corneal fibroblasts. PDGF AA, AB, and BB stimulated proliferation of human corneal fibroblasts. CONCLUSIONS: The PDGF growth factor receptor system is expressed in the human cornea. PDGF, BMP2, BMP4, and IL-1alpha may modulate keratocyte chemotaxis and proliferation during homeostasis and wound healing.

Prostate cancer chemoprevention by green tea: in vitro and in vivo inhibition of testosterone-mediated induction of ornithine decarboxylase.

Recently, we have shown that ornithine decarboxylase (ODC), a rate-controlling enzyme in the polyamine biosynthetic pathway, is overexpressed in prostate cancer (PCA) and prostatic fluid in humans (R. R. Mohan et al., Clin. Cancer Res., 5: 143-147, 1999). ODC is also characterized as an androgen-responsive gene, and the androgenic stimulation regulates the development and growth of both normal and tumorigenic prostate cells. Thus, chemopreventive approaches aimed toward the modulation of ODC could be effective against PCA. Green tea polyphenols (GTPs) possess strong chemopreventive properties against a variety of animal tumor models and in some human epidemiological studies. At least two epidemiological studies have suggested that people who consume tea regularly may have a decreased risk of PCA. In this study, we investigated the effect of GTPs against testosterone-mediated induction of ODC in human prostate carcinoma cells, LNCaP as an in vitro model, and in Cpb:WU rats and C57BL/6 mice as in vivo models. Treatment of LNCaP cells with testosterone resulted in induction of ODC activity in a dose-dependent manner. Pretreatment of the cells with GTPs resulted in a significant inhibition of testosterone-caused induction of ODC activity in a dose-dependent manner. Similar effects of GTPs were observed in anchorage-independent growth assay of LNCaP cells where pretreatment of the cells with GTP was found to result in dose-dependent inhibition of colony formation. Testosterone treatment of the cells resulted in a significant increase in the level of ODC mRNA, and this increase was almost completely abolished by prior treatment of the cells with GTPs. The administration of testosterone (10 mg/kg body weight, i.p.) to sham-operated and castrated Cpb:WU rats resulted in 2- and 38-fold increases in ODC activity, respectively, in the ventral prostate. Oral feeding of 0.2% GTPs in drinking water for 7 days before testosterone administration resulted in 20 and 54% decreases in testosterone-caused induction of ODC activity in sham-operated and castrated rats, respectively. Similar results were obtained with C57BL/6 mice, where testosterone treatment at similar dosage resulted in a 2-fold increase in ODC activity in the ventral prostate and prior oral feeding with 0.2% GTPs resulted in 40% inhibition in this induction.

Stromal-epithelial interactions in the cornea.

Stromal-epithelial interactions are key determinants of corneal function. Bi-directional communications occur in a highly coordinated manner between these corneal tissues during normal development, homeostasis, and wound healing. The best characterized stromal to epithelial interactions in the cornea are mediated by the classical paracrine mediators hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF). HGF and KGF are produced by the keratocytes to regulate proliferation, motility, differentiation, and possibly other functions, of epithelial cells. Other cytokines produced by keratocytes may also contribute to these interactions. Epithelial to stromal interactions are mediated by cytokines, such as interleukin-1 (IL-1) and soluble Fas ligand, that are released by corneal epithelial cells in response to injury. Other, yet to be identified, cytokine systems may be released from the unwounded corneal epithelium to regulate keratocyte viability and function. IL-1 appears to be a master regulator of corneal wound healing that modulates functions such as matrix metalloproteinase production, HGF and KGF production, and apoptosis of keratocyte cells following injury. The Fas/Fas ligand system has been shown to contribute to the immune privileged status of the cornea. However, this cytokine-receptor system probably also modulates corneal cell apoptosis following infection by viruses such as herpes simplex and wounding. Pharmacologic control of stromal-epithelial interactions appears to offer the potential to regulate corneal wound healing and, possibly, treat corneal diseases in which these interactions have a central role.

Expression of HGF, KGF, EGF and receptor messenger RNAs following corneal epithelial wounding.

Hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), epidermal growth factor (EGF), and their receptors have been associated with homeostasis and wound healing in the cornea. The purpose of this study was to examine the expression of the messenger RNAs for these growth factors and receptors in a wounded series of mouse corneas using in situ hybridization. In situ hybridization was performed with 3H-labeled riboprobes on unwounded corneas and corneas at 30 minutes, 4, 12, 24, 48 and 72 hr, and 7 days after epithelial scrape wounds in Balb/C mice. Qualitative and semi-quantitative analyses were performed. Expression of HGF, KGF and EGF mRNAs in keratocytes in the unwounded cornea was low. EGF mRNA was also expressed in unwounded corneal epithelium. Following wounding, however, these growth factor mRNAs were markedly upregulated in keratocytes. EGF mRNA expression in the epithelium appeared unaffected by wounding. At seven days after wounding and several days following closure of the epithelial defect, HGF mRNA and KGF mRNA were still expressed at higher levels in keratocytes compared with unwounded corneas. No difference in expression of HGF or KGF mRNAs between limbal, peripheral corneal, or central corneal keratocytes was noted in the unwounded cornea, KGF receptor mRNA was prominently expressed throughout the unwounded corneal epithelium. HGF receptor mRNA and EGF receptor mRNAs were expressed at low levels in unwounded cornea epithelium. Following scrape injury, expression of HGF receptor mRNA and KGF receptor mRNA were markedly upregulated in the corneal epithelium, while no significant increase in EGF receptor mRNA expression was noted. These studies suggest a prominent role for HGF and KGF in modulating corneal epithelial wound healing following injury. Less prominent changes in EGF mRNA and EGF receptor mRNA in the corneal epithelium following wounding may suggest that EGF has more of a role in homeostasis in the mouse corneal epithelium.

A flavonoid antioxidant, silymarin, affords exceptionally high protection against tumor promotion in the SENCAR mouse skin tumorigenesis model.

In cancer chemoprevention studies, the identification of better antitumor-promoting agents is highly desired because they may have a wider applicability against the development of clinical cancers. Both epidemiological and animal studies have suggested that microchemicals present in the diet and several herbs and plants with diversified pharmacological properties are useful agents for the prevention of a wide variety of human cancers. Silymarin, a flavonoid isolated from milk thistle, is used clinically in Europe and Asia as an antihepatotoxic agent, largely due to its strong antioxidant activity. Because most antioxidants afford protection against tumor promotion, in this study, we assessed the protective effect of silymarin on tumor promotion in the SENCAR mouse skin tumorigenesis model. Application of silymarin prior to each 12-O-tetradecanoylphorbol 13-acetate (TPA) application resulted in a highly significant protection against tumor promotion in 7,12-dimethylbenz(a)anthracene-initiated mouse skin. The protective effect of silymarin was evident in terms of reduction in tumor incidence (25, 40, and 75% protection, P < 0.001, X2 test), tumor multiplicity (76, 84, and 97% protection, P < 0.001, Wilcoxon rank sum test), and tumor volume (76, 94, and 96% protection, P < 0.001, Student's t test) at the doses of 3, 6, and 12 mg per application, respectively. To dissect out the stage specificity of silymarin against tumor promotion, we next assessed its effect against both stage I and stage II of tumor promotion. Application of silymarin prior to that of TPA in stage I or mezerein in stage II tumor promotion in dimethylbenz(a)anthracene-initiated SENCAR mouse skin resulted in an exceptionally high protective effect during stage I tumor promotion, showing 74% protection against tumor incidence (P < 0.001, X2 test), 92% protection against tumor multiplicity (P < 0.001, Wilcoxon rank sum test), and 96% protection against tumor volume (P < 0.001, Student's t test). With regard to stage II tumor promotion, silymarin showed 26, 63, and 54% protection in tumor incidence, multiplicity, and volume, respectively. Similar effect of silymarin to that in anti-stage I studies, were also observed when applied during both stage I and stage II protocols. In other studies, silymarin significantly inhibited: (a) TPA-induced skin edema, epidermal hyperplasia, and proliferating cell nuclear antigen-positive cells; (b) DNA synthesis; and (c) epidermal lipid peroxidation, the early markers of TPA-caused changes that are associated with tumor promotion. Taken together, these results suggest that silymarin possesses exceptionally high protective effects against tumor promotion, primarily targeted against stage I tumors, and that the mechanism of such effects may involve inhibition of promoter-induced edema, hyperplasia, proliferation index, and oxidant state.

Changes in corneal morphology associated with chronic epithelial injury.

PURPOSE: The purpose of this study was to evaluate the effect of chronic epithelial scrape injury on corneal morphology. METHODS: The corneal epithelia in one eye of 8-week-old New Zealand White rabbits were scraped at weekly intervals. Central corneal thickness was measured by ultrasonic pachymetry before epithelial scrape each week. Control never wounded (C), chronic wounded with scrape the last week (W), and chronic wounded without scrape the last week (WW) corneas were processed for histologic analysis and transmission electron microscopy (TEM). The time intervals for histologic analysis were 4 (4 C, 2 W, 2 WW), 8 (4 C, 2 W, 2 WW), and 16 (7 C, 2 W, 5 WW) weeks. Histologic findings were monitored using hematoxylin and eosin staining, the TdT-dUTP terminal nick-end labeling (TUNEL) assay, and TEM. RESULTS: Chronic wounded corneas developed marked epithelial hyperplasia and a subepithelial acellular zone. Keratocytes undergoing apoptosis were primarily detected adjacent to the acellular zone by TUNEL assay and TEM. Total central corneal thickness measured by ultrasonic pachymetry (n=7) was significantly thinner in chronically scraped eyes compared with control eyes after 8, 12, and 16 weeks (P < 0.05). Control corneas increased in total thickness over the 16 weeks of the study, but there was no significant change in total thickness of the corneas that had chronic epithelial scrape injury over this time interval. Two scraped corneas had marked decreases in total corneal thickness relative to the corneal thickness at the beginning of the study. Epithelial hyperplasia developed in all scraped corneas examined histologically after 4, 8, or 16 weeks of scraping. When central epithelial thickness measured on hematoxylin and eosin-stained sections was subtracted from the total pachymetric corneal thickness to give approximate stromal thickness, the stromal thickness was 23% lower in the chronic wounded (277+/-15 microm) compared with the unwounded (356+/-6 microm) corneas (P=0.0008) after 16 weeks of wounding. CONCLUSIONS: Chronic epithelial injury induces stromal thinning and epithelial hyperplasia. These changes in cornea structure associated with chronic epithelial injury may have relevance to the pathophysiology of keratoconus.

Overexpression of ornithine decarboxylase in prostate cancer and prostatic fluid in humans.

Prostate cancer (PCA), the most commonly diagnosed cancer in males in the United States, is the second leading cause of cancer-related deaths of males in this country. Because of the poor success rate in the treatment of PCA, an intervention at an early stage may reduce the progression of small carcinoma to large metastatic lesion, thereby reducing PCA-related deaths. Concerted efforts are needed to establish mechanism-based approaches to develop: (a) the markers for early detection of the disease as well as toward monitoring the efficacy of treatment(s); and (b) novel chemopreventive strategies against PCA. Using unique samples of pair-matched benign and cancer tissue obtained from the same PCA patient, we showed that ornithine decarboxylase (ODC) activity is significantly (P < 0.001) elevated in PCA (1142 +/- 100; mean +/- SE) than in paired benign tissue (427 +/- 51; mean +/- SE). The immunoblot analysis also showed a significant elevation in the protein expression of ODC in the PCA tissues as compared with the paired benign tissue. Furthermore, our data showed that the ODC activity in the prostatic fluid obtained by a digital rectal massage from the patients with PCA (3847 +/- 162; mean +/- SE) was significantly higher than in the patients with benign prostatic hyperplasia (2742 +/- 167; mean +/- SE) or normal individuals (1244 +/- 67; mean +/- SE). This observation might be of significance because the prostatic fluid could be obtained noninvasively by digital rectal massage. We suggest that ODC could serve as a target for early detection of human PCA as well as for monitoring the efficacy of treatment(s). The development of ODC as a target for novel chemopreventive strategies against PCA is an intriguing possibility.

Bone morphogenic proteins 2 and 4 and their receptors in the adult human cornea.

PURPOSE: To examine the expression of transforming growth factor family members bone morphogenic proteins 2 and 4 (BMP2, BMP4), their receptor mRNAs, and BMP2 and BMP4 proteins in the cells of the human cornea. The effects of BMP2 and BMP4 on corneal fibroblast proliferation and apoptosis were also examined. METHODS: Reverse transcription-polymerase chain reaction, immunoprecipitation, and western blot analysis were used to examine mRNA and protein expression in cultured human corneal cells. Immunocytochemistry was applied to examine protein localization in fresh frozen human cornea cells. Stimulation and inhibition of nuclear factor-kappaB (NF-kappaB) activation was evaluated by gel shift assay. Apoptosis was examined using trypan blue exclusion, laddering of DNA, CPP32 assay, and transmission electron microscopy. Proliferation was monitored by counting cells. RESULTS: BMP2 and BMP4 mRNAs and proteins were expressed in cultured human corneal epithelial cells, keratocytes, and corneal endothelial cells. BMP2 and BMP4 were detected in each major corneal cell type in fresh frozen human cornea. BMP receptor IA, IB, and II mRNAs were also detected in cultured human corneal epithelial cells, keratocytes, and endothelial cells. BMP2 and BMP4 stimulated activation of NF-kappaB. Actinomycin D and SN50 peptide, but not SN50M control peptide, inhibited NF-kappaB activation in response to BMP2 or BMP4. BMP2 and BMP4 stimulated apoptosis of corneal fibroblast cells when NF-kappaB activation was inhibited with the nonselective inhibitor actinomycin D or selective inhibitor SN50. The nonsteroidal anti-inflammatory agents ketorolac tromethamine and diclofenac sodium augmented the effect of BMP2 on corneal fibroblast apoptosis. BMP2 and BMP4 both stimulated proliferation of corneal fibroblast cells in the absence of inhibitors of NF-kappaB activation. CONCLUSIONS: BMP2, BMP4, and their receptors are expressed in the cells of the adult human cornea. The functions regulated by these cytokines may include keratocyte proliferation and apoptosis.