Quantitative analysis of p16 methylation in Barrett's carcinogenesis.

p16 hypermethylation in Barrett's carcinogenesis has been evaluated in studies which did not take into account sample heterogeneity and yielded qualitative (methylated/unmethylated) instead of accurate quantitative (percentage of CpG methylation) data. We aimed to measure the degree of p16 methylation in pure samples representing all the steps of Barrett's tumorogenesis and to evaluate the influence of sample heterogeneity in methylation analysis. METHODS: 77 paraffin-embedded human esophageal samples were analyzed. Histological grading was established by two pathologists in: negative for dysplasia, indefinite for dysplasia, low-grade dysplasia, high-grade dysplasia and adenocarcinoma. Areas of interest were selected by laser-capture microdissection. p16 methylation was quantified by pyrosequencing. An adjacent section of the whole sample was also analyzed to compare methylation data. RESULTS: After microdissection, we obtained 15 samples of squamous epithelium, 36 non-dysplastic Barrett's esophagus, 3 indefinite for dysplasia, 24 low-grade dysplasia, 4 high-grade dysplasia and 12 adenocarcinoma. Squamous epithelium showed the lowest methylation rates: 6% (IQR 5-11) vs. 11%(7-39.50) in negative/indefinite for dysplasia, p<0.01; 10.60%(6-24) in low-grade dysplasia, p<0.05; and 44.50%(9-66.75) in high-grade dysplasia/adenocarcinoma, p<0.01. This latter group also exhibited higher methylation rates than Barrett's epithelium with and without low-grade dysplasia (p<0.05). p16 methylation rates of microdissected and non-microdissected samples did not correlate unless the considered histological alteration comprised >71% of the sample. CONCLUSIONS: p16 methylation is an early event in Barrett's carcinogenesis which increases with the severity of histological alteration. p16 methylation rates are profoundly influenced by sample heterogeneity, so selection of samples is crucial in order to detect differences.

Low-Dose Aspirin Acetylates Cyclooxygenase-1 in Human Colorectal Mucosa: Implications for the Chemoprevention of Colorectal Cancer.

The mechanism of action of low-dose aspirin in the prevention of colorectal cancer (CRC) remains largely hypothetical. We aimed to compare the effects of low-dose aspirin (100 mg/day for 7 days) given to 40 individuals undergoing CRC screening on the extent of cyclooxygenase (COX)-1 acetylation at serine-529 (AceCOX-1), in blood platelets vs. colorectal mucosa, at 7 (group 1) and 24 h (group 2) after dosing. A significantly (P < 0.01) lower %AceCOX-1 was detected in colonic and rectal mucosa (average 64%) vs. platelets (average 75%) in both groups. This effect was associated with an average 46% (P < 0.01) and 35% (P < 0.05) reduction in prostaglandin (PG) E2 levels and phosphorylated S6 (p-S6) levels, respectively. Rectal mucosal levels of p-S6/S6 significantly (P < 0.01) correlated with PGE2 . These findings demonstrate that low-dose aspirin produces long-lasting acetylation of COX-1 and downregulation of p-S6 in human colorectal mucosa, an effect that may interfere with early colorectal carcinogenesis.

Reappraisal of the clinical pharmacology of low-dose aspirin by comparing novel direct and traditional indirect biomarkers of drug action.

BACKGROUND: Even though the acetylation of platelet cyclooxygenase (COX)-1 at serine-529 is the direct mechanism of action of low-dose aspirin, its antiplatelet effect has been characterized using indirect indexes of COX-1 activity. OBJECTIVES: We performed a clinical study with enteric-coated low-dose aspirin (EC-aspirin), in healthy subjects, to evaluate the effects on the extent and duration of platelet COX-1 acetylation, using a novel proteomic strategy for absolute protein quantification (termed AQUA), as compared with traditional pharmacokinetic and pharmacodynamic parameters. SUBJECTS AND METHODS: In a phase I, single-arm, open-label study of EC aspirin (100 mg day(-1) ) administered to 24 healthy subjects, we compared, over a 24 h-period on day 1 and 7, % platelet acetylated COX-1 (AceCOX-1) with traditional pharmacokinetic and pharmacodynamics [i.e. serum thromboxane (TX) B2 , platelet function by monitoring CEPI(collagen/epinephrine) closure time (CT) using whole-blood PFA-100 and urinary excretion of 11-dehydro-TXB2 ] parameters. RESULTS: Acetylation of platelet COX-1 was measurable before detection of aspirin levels in the systemic circulation and increased in a cumulative fashion upon repeated dosing. After the last dose of EC-aspirin, %AceCOX-1, serum TXB2 and CEPI-CT values were maximally and persistently modified throughout 24 h; they averaged 76 ± 2%, 99.0 ± 0.4% and 271 ± 5 s, respectively. EC-aspirin caused 75% reduction in urinary 11-dehydro-TXB2 excretion. After chronic dosing with aspirin, the pharmacokinetics of acetylsalicylic acid was completely dissociated from pharmacodynamics. CONCLUSIONS: The demonstrated feasibility of quantifying the extent and duration of platelet COX-1 acetylation will allow characterizing the genetic, pharmacokinetic and pharmacodynamic determinants of the inter-individual variability in the antiplatelet response to low-dose aspirin as well as identifying extra-platelet sites of drug action.

In vitro vitamin K3 effect on conjunctival fibroblast migration and proliferation.

PURPOSE: To evaluate the dose effect of vitamin K3 on wound healing mechanisms. METHODS: Conjunctival fibroblasts were incubated for 24 hours. An artificial wound was made and the cells were incubated with fresh medium plus doses of vitamin K3 to be tested. Wound repair was monitored at 0, 18, 24, and 48 hours. Proliferation was measured in actively dividing cells by [(3)H]thymidine uptake. Six different groups were tested: group 1/no drugs added, group 2/ethanol 0.1%, group 3/vitamin K3 1 mg/L, group 4/vitamin K3 2 mg/L, group 5/vitamin K3 4 mg/L, and group 6/vitamin K3 6 mg/L. Each experiment was carried out in triplicate and 4 times. RESULTS: There were no differences among groups at the initial time. In vitro wound repair was slower in groups 4, 5, and 6. There were no differences between control and ethanol groups and between control and vitamin K3 1 mg/L groups. Fibroblast mitogenic activity was statistically decreased in all vitamin K groups; statistical differences were found among vitamin K3 1 mg/mL and higher doses too. In groups 5 and 6, cellular toxicity was presented. CONCLUSIONS: Vitamin K3 is able to inhibit fibroblast proliferation. Vitamin K3 2 mg/L or higher doses inhibit wound healing repair, exhibiting cellular toxicity at 4 and 6 mg/L.

Characterization of the prostaglandin E2 pathway in a rat model of esophageal adenocarcinoma.

UNLABELLED: Accumulating evidence indicates that the cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) pathway plays a key role in esophageal carcinogenesis. A better understanding of the pathway downstream of COX-2 may reveal novel targets for the prevention of esophageal adenocarcinoma (EAC). The objective of this study was to characterize the profile of genes involved in PGE2 metabolism and signaling in an experimental model of EAC. Esophagojejunostomy with gastric preservation was performed in wistar rats to induce gastroduodenal reflux. Rats were sacrificed 2 or 4 months after surgery. Nine non-operated rats were used to obtain normal (control) esophageal tissues. RESULTS: All rats that underwent esophagojejunostomy developed inflammation. In addition, 90% of the animals showed intestinal metaplasia; of those, 40% progressed to AC. This process was accompanied by a significant increase in esophageal PGE2 levels and the induction of both mRNA and protein levels of COX-2, COX-1, prostaglandin E synthase, 15-hydroxyprostaglandin dehydrogenase, and PGE2 receptors EP3, EP4 and especially EP2, which rose to particularly high levels in experimental rats. In addition, exposure to a selective COX-2 inhibitor (SC58125) or an EP1/EP2 antagonist (AH6809), but not an EP4 antagonist (AH23848B), significantly reduced cell proliferation of esophageal explants in 24 hour-organ culture experiments. Our data suggest that, in addition to COX-2, other components of the PGE2 pathway, including COX-1, may play important roles in the development of EAC induced by gastroduodenal reflux in the rat. Although it must be confirmed in vivo, the EP2 receptor may represent a promising selective target in the prevention of Barrett's associated AC.

Cystatin C level as a predictor of death and cardiovascular events after liver transplantation.

BACKGROUND: The diagnosis of renal failure is important in cirrhosis. Cystatin C (Cys) has been suggested not only to be a sensitive marker of renal function, but also a stronger predictor of the risk of death and cardiovascular events in heart failure. Our aims were to investigate plasma Cys concentrations for the detection of moderately impaired renal function and its association with mortality and cardiovascular events among cirrhotic patients after liver transplantation (OLT). METHODS: Clinical and biochemical data, including Cys levels, were analyzed in 99 cirrhotic candidates for OLT. We recorded cardiovascular events. RESULTS: Receiver-operator characteristics curves showed a similar efficiency to detect a creatinine clearance <60 mL/min per 1.73m(2) (Cys = 0.753; creatinine [Cr] = 0.799; glomerular filtration rate [GFR, Cockcroft-Gault formula] = 0.842; urea = 0.823; P = .001). However, at cutoff concentrations of 1.3 mg/dL, Cr showed great specificity (96%) but poor sensitivity (13%), while the sensitivity of Cys was superior (83%) with moderate specificity (55%) at a cutoff of 1400 ng/mL. Over a median follow-up of 2.7 years, 14 patients developed a cardiovascular event, including, 11 who displayed Cys levels >1400 ng/mL before OLT, showing a significant difference (P < .05) compared to patients who showed no cardiovascular event. Kaplan-Meier analysis Cys discriminated significantly better than the Model for End-Stage Liver Disease score between survivors and nonsurvivors (P < .05). CONCLUSION: Cys determinations could be a valuable tool for early diagnosis of renal dysfunction among cirrhotic patients. Furthermore, it may predict the risk of death and cardiovascular events after OLT.

Prostaglandin EP2 receptor expression is increased in Barrett's oesophagus and oesophageal adenocarcinoma.

BACKGROUND: Accumulating evidence suggests that cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) is involved in oesophageal adenocarcinogenesis. PGE2 exerts its biological action by binding to specific receptors (EP1, EP2, EP3 and EP4). AIM: To investigate which PGE2 receptor subtypes regulate PGE2 signals in the oesophageal adenocarcinoma sequence. METHODS: Expression was determined in oesophageal biopsies from 85 patients with oesophagitis, Barrett's metaplasia, intraepithelial neoplasia, oesophageal adenocarcinoma and normal oesophagus. Levels of mRNA and protein expression were determined by quantitative PCR, immunohistochemistry and western-blot. Expression of EP receptors was also determined in response to acid and bile exposure in the Barrett's adenocarcinoma cell line OE33. RESULTS: All four EP receptors subtypes were expressed in human oesophageal tissues. COX-2 and, especially, EP2 were increased in the Barrett's metaplasia-intraepithelial neoplasia-adenocarcinoma sequence. Expression of the EP4 receptor protein was increased in oesophageal adenocarcinoma. In contrast, expression levels of COX-1 and EP3 receptor were decreased along the sequence. No differences in EP1 expression were found. Treatment with the bile acid deoxycholate increased COX-2, EP1, EP2 and EP4 expression in OE33 cells. CONCLUSIONS: Our data suggest that in addition to COX-2, EP2 and EP4 receptors could be a selective target in the prevention and/or treatment of the Barrett's-associated adenocarcinoma.

Effects of long-term cyclo-oxygenase 2 selective and acid inhibition on Barrett's oesophagus.

BACKGROUND: There is an overexpression of cyclo-oxygenase 2 (COX-2) in Barrett's oesophagus (BO). AIM: To determine the long-term effect of a COX-2 inhibitor on cellular mechanisms involved in BO. METHODS: A randomized controlled trial was conducted in BO patients allocated to continue the usual proton pump inhibitor (PPI) alone treatment, or PPI combined with rofecoxib (25 mg/day) for 6 months. Cell proliferation index and COX-2 expression in BO glands was determined in biopsy specimens at baseline and after treatment. Cell apoptosis, cyclin D1, p53 and vascular endothelial growth factor (VEGF) expression was also explored in a subset of patients. Student-t test and the U-Mann-Whitney test were used for quantitative and ordinal variables. RESULTS: Of 62 patients, 58 completed the study. A higher proportion of patients on rofecoxib + PPI exhibited a decrease in COX-2 expression compared to those treated with PPI alone, but cell proliferation index was not affected. Unlike PPI alone, rofecoxib + PPI was associated with an increase in the apoptotic cell index, a decrease in p53 cell staining and VEGF expression in mucosal vessels. No effect on low-grade dysplasia or cyclin D1 was observed. CONCLUSIONS: The addition of rofecoxib to PPI therapy does not affect cell proliferation index in BO cells after 6 months of therapy, but does reduce COX-2 and VEGF expression and increases cell apoptosis.

Investigación translacional en patología digestiva: del laboratorio a la clínica y viceversa beneficiario: el paciente / No disponible

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TGFB1 gene polymorphisms: their relevance in the susceptibility to Helicobacter pylori-related diseases.

Recent studies have revealed elevated expression of transforming growth factor beta1 (TGF-beta1) in gastric mucosa of patients with gastric cancer (GC) and those undergoing ulcer repair. As production of TGF-beta1 is genetically regulated, we aimed to assess whether functional polymorphisms of the TGFB1 gene are involved in susceptibility to and clinical characteristics of Helicobacter pylori-related diseases. DNA from 142 unrelated Spanish patients with GC, 200 with peptic ulcer and 342 healthy controls was typed for the MspA1I T+869C, and the Sau96I G+915C polymorphisms of the TGFB1 gene using polymerase chain reaction and RFLP analysis. H. pylori infection and CagA/VacA antibody status were determined by Western blot in patients and controls. H. pylori infection (odds ratio (OR): 11.44; 95% confidence interval (CI): 4.45-29.42; P<0.001) and non-steroidal anti-inflammatory drugs (OR: 5.07; 95% CI: 2.53-10.16; P<0.001) were identified as independent risks factors for duodenal ulcer (DU), whereas the TGFB1+869(*)C/C genotype was associated with reduced risk of developing the disease (OR: 0.32; 95% CI=0.15-0.68; P=0.003). Our results show that the TGFB1 T+869C gene polymorphism is involved in the susceptibility to DU and provide further evidence that host genetic factors play a key role in the pathogenesis of H. pylori-related diseases.

COX-2 inhibition in esophagitis, Barrett's esophagus and esophageal cancer.

There is extensive evidence that cyclooxygenase-2 (COX-2) plays a significant role in the process of carcinogenesis in different tumors. Although most of these evidences derive from studies in colorectal cancer, data obtained from recent studies strongly suggest that COX-2 might play an important role in the neoplastic transformation of esophageal epithelium. NSAIDs use is associated with a reduction of the risk of developing esophageal cancer, including adenocarcinoma. Up-regulation of COX-2 has been reported in different stages of the carcinogenic sequence leading to esophageal adenocarcinoma. Treatment with selective COX-2 inhibitors has been shown to reduce the damage induced by acid and pepsin in the esophageal mucosa of rabbits, the incidence of tumors in an animal model of esophageal adenocarcinoma and to decrease proliferation and induce apoptosis in both Barrett's epithelial and adenocarcinoma cells. The first clinical study has shown that selective inhibition of COX-2 is followed by a significant decrease of cell proliferation in human Barrett's metaplasia. Clinical trials have begun in order to assess the efficacy of selective COX-2 inhibitors to prevent the progression of Barrett's esophagus to adenocarcinoma. Bile salts and acid are likely to early induce COX-2 in this sequence, although other factors, such as proinflammatory cytokines, inducible nitric oxide synthase and growth factors such as TGF-beta, are potential COX-2 inducers in the esophagus. Further studies are necessary in order to better understand factors involved in COX-2 up-regulation and mechanisms of COX-2 associated tumorigenesis in the esophagus.

[CagA and VacA cytotoxin antibodies and risk for peptic ulcer disease in patients with Helicobacter pylori infection]. / Anticuerpos frente a las citotoxinas CagA y VacA y riesgo de úlcera péptica en pacientes con infección por Helicobacter pylori.

BACKGROUND: The presence of cagA antibodies constitutes a serum marker of infection caused by virulent strains of Helicobacter pylori. The objective of this study was to determine the risk of peptic ulcer in patients with H. pylori infection, in relation to the detection of CagA and VacA antibodies. PATIENTS AND METHOD: Prospective case-control study including 104 peptic ulcer patients with active H. pylori infection (positive urease test and/or histology, or positive urea breath test) and 104 age- and sex-matched controls, without peptic ulcer history, with active H. pylori infection (positive urea breath test). Serum CagA and VacA antibodies were determined by Western blot. Non-steroidal antiinflamatory drugs (NSAID) use was determined by structured data collection. A multivariate analysis (logistic regression) was carried out to determine the odds ratio (OR). RESULTS: Presence of serum antibodies against CagA was higher in peptic ulcer patients (74%) than in controls (46.2%) (OR = 5.7; 95% CI = 2.1-15.6). However, presence of serum antibodies against VacA in patients (46.2%) was similar to that in controls (36.5%). NSAID use was also more frequent in patients (51.9%) than in controls (21,2%) (OR = 6.5; 95% CI = 2.2-19,5). CONCLUSIONS: Serum antibodies against CagA and use of NSAID are the most important risk factors for peptic ulcer disease.

[Helicobacter pylori and immunogenetic factors of the host: relevance of the HLADQA1 *0102 and *0301 alleles in peptic ulcer]. / Helicobacter pylori y factores inmunogenéticos del huésped: relevancia de los alelos HLADQA1 *0102 y *0301 en la úlcera péptica.

AIM: To investigate the potential contribution of the *0102 and *0301 alleles of the HLADQA1 gene in Helicobacter pylori infection and peptic ulcer disease in a Spanish Caucasian population. PATIENTS AND METHODS: We studied 163 patients with peptic ulcer (117 duodenal ulcers and 46 gastric ulcers; 111 with recent upper gastrointestinal hemorrhage) and 90 controls. The *0102 and *0301 alleles of the HLADQA1 gene were typed by polymerase chain reaction using genomic DNA. H. pylori infection were determined by breath test and/or serology. The cytotoxins CagA and VacA were investigated using serology (Western-blot) in 98 patients and 48 controls with H. pylori infection. RESULTS: H. pylori infection was found in 94.6% of patients with duodenal ulcer, in 84.4% of those with gastric ulcer and in 67.4% of controls (p < 0.001). The distribution of the *0102 allele of the HLADQA1 gene was similar in patients (31.9%) and in controls (36.7%). The *0301 was more frequent in patients with gastric ulcer (32.6%) than in those with duodenal ulcer (16.2%) (p < 0.05) but no differences were found on comparison with the control group (24.4%). No differences were found when the groups were analyzed according to H. pylori infection, CagA- and VacA-positive strains, consumption of non-steroidal antiinflammatory drugs or previous history of ulcer or hemorrhage. CONCLUSION: The *0102 and *0301 alleles of the HLADQA1 gene did not alter susceptibility to H. pylori infection or the evolution of peptic ulcer disease in a Caucasian population in Spain.

Superoxide anion and nitric oxide in high-grade esophagitis induced by acid and pepsin in rabbits.

It has been proposed that free radicals are involved in the pathogenesis of esophageal mucosal damage induced by acid and pepsin. Recent data have suggested that nitric oxide (NO) is involved in the mucosal defense of the esophagus and that superoxide anion plays a minor role in low-grade esophagitis. To study the role and potential interaction of NO and superoxide anion in an experimental model of high-grade esophagitis, acidified pepsin was perfused (45 min/12 hr) for five days in rabbits with different agents to modulate the generation of these radicals. Measurements included both macroscopic and microscopic mucosal damage, superoxide anion generation, NO synthase mucosal activity, and peroxynitrite formation. High-grade esophagitis was associated with mucosal superoxide anion generation. Treatment with exogenous superoxide dismutase completely prevented mucosal damage. The perfusion of acidified pepsin in the lumen of the esophagus was initially associated with increased NO synthase mucosal activity but decreased with the progression of damage. Generation of peroxynitrites was present in those cases with severe damage. Treatment with NO-modifying agents did not induce consistent modification of mucosal damage. It is concluded that superoxide anion is involved in the induction of high-grade esophagitis and that it interacts with nitric oxide to generate peroxynitrite radicals in this model. Superoxide dismutase but not NO-donor-modifying agents might have a therapeutic role in preventing severe esophageal mucosal damage induced by acid and pepsin.

Platelet-derived growth factor and epidermal growth factor play a major role in human colonic fibroblast repair activities.

BACKGROUND AND AIMS: Ulceration is a common feature of inflammatory bowel diseases, where subepithelial cell growth is frequently necessary for resolution. In order to further understand the role of colonic fibroblasts in this process, we have used an in vitro model of wound repair to study the response of human colonic fibroblasts to several growth factors expressed in colonic tissues. METHODS: Proliferation was determined by [(3)H]thymidine incorporation into DNA in subconfluent fibroblast cultures. In vitro wound repair was determined in confluent fibroblast monolayers after mechanical denudation. The presence of growth factors secreted by fibroblasts was studied in conditioned medium by heparin affinity chromatography and immunodetection with specific antibodies. RESULTS: Serum and platelet-derived growth factor (PDGF-BB) induced a dramatic increase in both colonic fibroblast proliferation and closure of wounded cell monolayers. Epidermal growth factor (EGF) stimulated both fibroblast activities, but the effect was less potent. However, colonic fibroblasts did not respond to transforming growth factor-beta(1). Conditioned medium stimulated fibroblast proliferation and wound repair activity, which was reverted by the addition of suramin. Furthermore, a PDGF-like factor was isolated from colonic fibroblast-conditioned medium. CONCLUSIONS: EGF and PDGF-BB promote human colonic fibroblast-dependent wound repair activities. Human colonic fibroblasts may exert an autocrine regulation via the production of growth factors.

Efecto inhibitorio del nicardipino sobre los mecanismos de proliferación fibroblásticos / Inhibitory effect of nicardipine on fibroblast wound healing

Objetivo: Evaluar el efecto de distintas dosis del bloqueador de los canales del calcio nicardipino (NCP) sobre la migración y proliferación fibroblástica y su toxicidad celular. Métodos: Se realizó una úlcera mecánica sobre una monocapa confluente de fibroblastos de origen conjuntival. Una vez realizada la úlcera, se añadió al medio de cultivo la sustancia a evaluar. La capacidad de reparación se cuantificó midiendo el área que permanecía libre de fibroblastos a las 0, 18, 24 y 48 horas. Grupos de estudio: Grupo 1, grupo control. Grupo 2, se añadió NCP 10-4M disuelto en el medio de cultivo. Grupo 3, NCP 7,5x10-5M. Grupo 4, NCP 5x10-5M. Grupo 5, NCP 2,5x10-5M. Grupo 6, NCP 10-5M. Grupo 7, NCP 10-6M. Grupo 8, NCP 10-7M. Grupo 9, NCP 10-8M. Cada experimento se realizó por triplicado repitiéndose 4 veces. Resultados: El NCP a dosis de 5x10-5 y superiores inhibió el proceso de cicatrización en los tiempos 18, 24 y 48 horas. El crecimiento a las 48 horas con NCP a dosis 2,5x10-5M fue estadísticamente inferior al de los grupos control, grupo 7, 8 y 9. Con dosis iguales o superiores a 5x10-5 hubo atipias y muerte celular. Conclusiones: El NCP inhibe el proceso de reparación fibroblástica exhibiendo toxicidad en dosis iguales o superiores a 5x10-5M (AU)

Efecto del succinato ácido de alfa-tocoferol sobre los mecanismos de cicatrización fibroblásticos / Inhibitory effect of alpha tocopherol succinate on fibroblast wound healing

Objetivo: Evaluar el efecto de distintas dosis de succinato ácido de alfa tocoferol (SAT) sobre la migración y proliferación fibroblástica. Métodos: Se realizó una úlcera mecánica sobre una monocapa confluente de fibroblastos de origen conjuntival que habían permanecido en medio deprivado de suero durante 24 horas. Una vez realizada la úlcera se añadió al medio de cultivo la sustancia a evaluar. La capacidad de reparación se cuantificó midiendo el área que permanecía libre de fibroblastos a las 0, 18, 24 y 48 horas. Grupos de estudio: Grupo 1: grupo control: se adicionó etanol 0,1 por ciento. Grupo 2: se añadió al medio SAT 25 µM previamente disuelto en etanol (concentración final de etanol 0,1 por ciento). Grupo 3: SAT 50 µM en etanol 0,1 por ciento. Cada experimento se realizó por triplicado repitiéndose 4 veces. Resultados: No existieron diferencias entre los grupos en las primeras 24 horas. A las 48 horas las lesiones del grupo control presentaban una superficie libre de células significativamente menor que la de los dos grupos de SAT. No se detectaron alteraciones celulares en ninguno de los grupos. Conclusiones: El SAT a dosis de 25 y 50 µM tiene un efecto inhibidor de la proliferación fibroblástica sin toxicidad celular (AU)

[Inhibitory effect of alpha tocopherol succinate on fibroblast wound healing]. / Efecto del succinato ácido de alfa-tocoferol sobre los mecanismos de cicatrización fibroblásticos.

PURPOSE: To evaluate and compare the effect of acid tocopherol succinate (ATS) on fibroblast migration and proliferation. METHODS: In vitro wound repair was determined in confluent fibroblast monolayer. Conjunctival fibroblasts were incubated with serum-deprived medium for 24 hours. After this time an artificial wound was made and the cells were incubated with fresh medium plus the doses of ATS to be tested. The cell free area was monitored at 0, 18, 24 and 48 hours. Groups of treatment: Group 1: ethanol 0.1%. Group 2: ATS 25 microM dissolved in ethanol (final concentration 0.1%) Group 3: ATS 50 microM in ethanol 0.1%. Each experiment was carried out in triplicate and repeated 4 times. RESULTS: There were no differences among the groups during the first 24 hours. ATS showed significantly larger cell-free area at 48 hours. There were no signs of cellular toxicity. CONCLUSIONS: 25 microM and 50 microM ATS inhibit fibroblast proliferation without cellular toxicity.

[Inhibitory effect of nicardipine on fibroblast proliferation mechanisms]. / Efecto inhibitorio del nicardipino sobre los mecanismos de proliferación fibroblásticos.

PURPOSE: To assess the effect of nicardipine (NCP) on fibroblast migration and proliferation, and its cellular toxicity. METHODS: In vitro wound repair was assessed in confluent fibroblast monolayer. Mechanical round wounds were performed in the monolayers and the cultures were incubated in fresh media plus NCP. The cell-free area was monitored after 0, 18, 24 and 48 hours. Groups of treatment: Group 1, Sham. Group 2, NCP 10(-4)M in the media. Group 3, NCP 7.5x10(-5)M. Group 4, NCP 5x10(-5)M. Group 5, NCP 2.5x10(-5)M. Group 6, NCP 10(-5)M. Group 7, NCP 10(-6)M. Group 8, NCP 10(-7)M. Group 9, NCP 10(-3)M. Each experiment consisted of three tests that were repeated four times. RESULTS: The fibroblast migration and proliferation was inhibited at 5x10(-5)M or higher doses. The proliferation after 48 hours with NCP 2.5x10(-5)M was statistically inferior to the control group and groups 7, 8, and 9. NCP 5x10(-5)M or higher doses showed cellular atypia and cell death. CONCLUSIONS: NCP effectively inhibits fibroblastic wound repair process at doses 2.5x10(-5)M and shows toxicity at doses over 5x10(-5)M.