Inhibiting PRMT1 protects against CoNV by regulating macrophages through the FGF2/PI3K/Akt pathway.

Corneal neovascularization (CoNV) is predominantly initiated by inflammatory processes, resulting in aberrant vascular proliferation and consequent visual impairment. Existing therapeutic interventions for CoNV demonstrate limited efficacy and potential for adverse reactions. Protein arginine methyltransferase 1 (PRMT1) is associated with the regulation of inflammation and M2 macrophage polarization. Nevertheless, the precise mechanism by which PRMT1 operates in CoNV remains uncertain. This study explored the impact of PRMT1 inhibition in a murine model of CoNV induced by alkali burn. Our findings indicated a direct relationship between PRMT1 levels and corneal damage. Moreover, our observations indicated an increase in fibroblast growth factor 2 (FGF2) expression in CoNV, which was reduced after treatment with a PRMT1 inhibitor. The inhibition of PRMT1 alleviated both corneal injury and CoNV, as evidenced by decreased corneal opacity and neovascularization. Immunofluorescence analysis and evaluation of inflammatory factor expression demonstrated that PRMT1 inhibition attenuated M2 macrophage polarization, a phenomenon that was reversed by the administration of recombinant FGF2 protein. These results were confirmed through experimentation on Human Umbilical Vein Endothelial Cells (HUVECs) and Mouse leukemia cells of monocyte macrophage cells (RAW264.7). Furthermore, it was established that FGF2 played a role in PI3K/Akt signal transduction, a critical regulatory pathway for M2 macrophage polarization. Importantly, the activity of this pathway was found to be suppressed by PRMT1 inhibitors. Mechanistically, PRMT1 was shown to promote M2 macrophage polarization, thereby contributing to CoNV, through the FGF2/PI3K/Akt pathway. Therefore, targeting PRMT1 may offer a promising therapeutic approach.

The potential protective effects of miR-497 on corneal neovascularization are mediated via macrophage through the IL-6/STAT3/VEGF signaling pathway.

Corneal neovascularization (CoNV) can cause abnormal blood vessels to grow in the transparent cornea, leading to various sight-threatening eye diseases. MicroRNAs are known to play essential roles in the regulation of numerous biological functions. We try to clarify the role of a specific microRNA, miR­497, which has been shown to regulate the growth of tumor cells and angiogenesis on the basis of available data. However, the association between miR-497 and vascularized cornea remains unclear. Therefore, it is urgently needed to understand the molecular mechanism of miR497 in the progress of corneal neovascularization. Animal model of CoNV was established in wildtype (WT) C57BL/6 mice, CRISPR/Cas9 mediated miR-497 knockout (KO) and overexpressed (TG) C57BL/6 mice. MiR-497, expressed in corneas, was actively involved in alkali burn-induced corneal neovascularization via targeting STAT3 and negatively regulating its expression, attenuating macrophage infiltration and M2 polarization. Knockdown of miR-497 enhanced the formation of corneal angiogenesis through targeting STAT3 and facilitating its expression, promoting recruitment of macrophages, while overexpression of miR-497 restrained blood vessel sprouting via regulating downstream STAT3 and VEGFA expression, reducing macrophage activation and inhibiting M2 polarization. Moreover, miR-497 knockout-mediated damage effect can be rescued through the inhibition of STAT3 signaling. Mechanically, miR-497 might serve as a potential strategy for pathological corneal neovascularization via macrophage through the IL-6/STAT3/VEGFA signaling pathway.

Paxillin promotes the migration and angiogenesis of HUVECs and affects angiogenesis in the mouse cornea.

Neonatal vascular ophthalmopathy is a refractory ophthalmologic disease, and is a major cause of blindness. Occurrence of neonatal vascular ophthalmopathy may be associated with Paxillin, a cellular adhesion molecule which promotes the migration of endothelial cells and angiogenesis. To explore the role of PXN in corneal angiogenesis, human umbilical vein endothelial cells were divided into five groups: i) Control group; ii) Empty vector-transfected control group; iii) PXN knockdown group (shPXN group); iv) PXN-negative control (NC) group; and v) PXN over-expressed group (overExp group). PXN protein levels, migration and tube formation were assessed in the different experimental groups. Mice were divided into four groups: i) Control; ii) Model; iii) shPXN; and iv) overExp groups. Tube formation was significantly increased in the overExp group compared with the empty vector-transfected control group (P<0.01). Tube formation was significantly decreased in the shPXN group compared with the PXN-NC group (P<0.01). In mice, blood corpuscles were significantly decreased in the shPXN group. PXN promoted the migration of endothelial cells and corneal angiogenesis. The results of the present study suggest a role for PXN in corneal angiogenesis and provide a theoretical basis and potential target for the treatment of corneal angiogenesis.

Inhibition of EZH2 alleviates angiogenesis in a model of corneal neovascularization by blocking FoxO3a-mediated oxidative stress.

Enhancer of zeste homolog 2 (EZH2), a well-known methyltransferase, mediates histone H3 lysine 27 trimethylation (H3K27me3) and plays a vital role in ophthalmological disease. However, its role in corneal neovascularization (CoNV) remains unclear. In vitro and in vivo models were assessed in hypoxia-stimulated angiogenesis and in a mouse model of alkali burn-induced CoNV. Human umbilical vein endothelial cells (HUVECs) were cultured under hypoxic conditions and different reoxygenation times to identify the molecular mechanisms involved in this process. In this study, we found that EZH2 was positively related to corneal alkali burn-induced injury. Inhibition of EZH2 with 3-Deazaneplanocin A (DZNeP) alleviated corneal injury, including oxidative stress and neovascularization in vivo. Similarly, inhibition of EZH2 with either DZNeP or small interfering RNA (siRNA) exerted an inhibitory effect on hypoxia/reoxygenation (H/R)-induced oxidative stress and angiogenesis in HUVECs. Moreover, our study revealed that ablation of reactive oxygen species (ROS) with N-acetyl-cysteine suppressed angiogenesis in HUVECs exposed to H/R stimulation. Furthermore, Forkhead-box protein O3a (FoxO3a), which was positively associated with ROS production and angiogenesis, was elevated during H/R. This effect could be reversed through the suppression of the transcription activity of EZH2 with DZNeP or siRNA. In addition, the PI3K/Akt pathway, which is the upstream of FoxO3a, was activated in both DZNeP-treated mice and EZH2-inhibited HUVECs. Collectively, our results demonstrated that the inhibition of EZH2 alleviated corneal angiogenesis by inhibiting FoxO3a-dependent ROS production through the PI3K/Akt signaling pathway. These findings indicate that EZH2 may be a valuable therapeutic target for CoNV.

Anomalous retinal artery associated with branch retinal artery occlusion and neovascular glaucoma: A case report.

BACKGROUND: Congenital anomalous retinal artery is rare and does not typically affect visual acuity. However, an abnormal artery that passes through and supplies blood to the macular area complicated with branch retinal artery occlusion may negatively impact visual acuity. This study reports an unusual case of anomalous retinal artery combined with retinal artery occlusion. CASE SUMMARY: A 52-year-old male presented with severely reduced vision in the right eye. The fundus examination revealed an anomalous artery, extending from the superior temporal arcade and crossing the macula into the inferior temporal quadrant. The anomalous artery was partially occluded, with a narrowed lumen. A cherry-red spot was observed with whitening of the macular area, suggesting macular edema. Fundus fluorescein angiography revealed disc leakage and a delayed filling time. Optical coherence tomography revealed increased thickness of the neuroretina and underlying layers. The patient was treated with vessel dilation, hyperbaric oxygen, ocular massage, and thrombolytics. Visual acuity of the right eye subsequently improved to 20/200 from hand motion at 4 cm. This improvement in visual acuity persisted when the patient was examined at the 1-mo follow-up visit. The patient was subsequently followed via telephone interview. The information provided via interview indicated that visual acuity in the affected eye was stable up to 6 years from the time of the initial presentation. However, after 3 additional years, the patient was diagnosed with neovascular glaucoma in the right eye, which was subsequently enucleated. CONCLUSION: Although congenital retinal vascular anomaly, including anomalous retinal artery, rarely affects vision, when complicated with branch retinal artery occlusion, the abnormal artery that supplies the macula may severely reduce visual acuity.

MFAP2 is overexpressed in gastric cancer and promotes motility via the MFAP2/integrin α5ß1/FAK/ERK pathway.

Gastric cancer (GC) is one of the most common malignancies and its prognosis is extremely poor. This study identifies a novel oncogene, microfibrillar-associated protein 2 (MFAP2) in GC. With integrative reanalysis of transcriptomic data, we found MFAP2 as a GC prognosis-related gene. And the aberrant expression of MFAP2 was explored in GC samples. Subsequent experiments indicated that silencing and exogenous MFAP2 could affect motility of cancer cells. The inhibition of silencing MFAP2 could be rescued by another FAK activator, fibronectin. This process is probably through affecting the activation of focal adhesion process via modulating ITGB1 and ITGA5. MFAP2 regulated integrin expression through ERK1/2 activation. Silencing MFAP2 by shRNA inhibited tumorigenicity and metastasis in nude mice. We also revealed that MFAP2 is a novel target of microRNA-29, and miR-29/MFAP2/integrin α5ß1/FAK/ERK1/2 could be an important oncogenic pathway in GC progression. In conclusion, our data identified MFAP2 as a novel oncogene in GC and revealed that miR-29/MFAP2/integrin α5ß1/FAK/ERK1/2 could be an important oncogenic pathway in GC progression.

Bilateral carotid-cavernous fistula with spontaneous resolution: A case report and literature review.

RATIONALE: Bilateral carotid-cavernous fistula (CCF) is rare and serious extra-ocular disease occurring in clinical which may result in severe complication. Unique manifestations and imaging examinations are important to the diagnosis. PATIENT CONCERNS: A case of bilateral carotid-cavernous fistula in an 60-year-old healthy man caused by a head injury is reported. Further clinical symptoms and signs and imaging examinations lead to the correct diagnosis. DIAGNOSES: Computed tomography angiography of the brain aroused suspicion of bilateral CCF. On physical examination, intraocular pressure in the right eye was 35 mm Hg, while the other eye was 56 mm Hg. INTERVENTIONS: After diagnosis, the patient chose conservative treatment for some reasons. OUTCOMES: The symptom of him had relieved in both eyes but no light perception in the right eye after two months telephone follow-up. LESSONS: Our case study demonstrated that a highly suspicion must be maintained when managing such patients to prevent serious consequences. At the same time, the early diagnosis and treatment of the disease have a critical relationship to the prognosis of patients, which should be paid attention to.

A meta-analysis of tobramycin and dexamethasone ointment in treatment of blepharitis / 国际眼科杂志(Guoji Yanke Zazhi)

AIM:To investigate and evaluate the effectiveness and safety of tobramycin and dexamethasone ointment in the treatment of blepharitis.METHODS:We searched in Pubmed,Medline,Embase,Elsevier,Cochrane Library,Wangfang database,CNKI,VIP database,Sinomed database,and gray literature were performing manual.The efficiency,intraocular pressure,adverse reactions and extract valid data were evaluated.RESULTS:Totally 8 controlled trials were enrolled,including 970 patients.Compared with control groups,the efficiency and the intraocular pressure in experiment group had a statistically significant increase (RR=1.75,95％ CI=1.29-2.37,P=0.0003;SMD=1.30,95％ CI=0.85-1.75,P＜0.00001),and there was no statistic difference in adverse reactions (RR =1.64,95％ CI =0.86-3.10,P =0.13).CONCLUSION:Tobramycin and dexamethasone ointment in treatment of blepharitis is effective with no adverse effects.Due to the quality of the literature is low,there is still need high quality randomized controlled trials.

Optical Quality and Related Factors in Ocular Hypertension: Preliminary Study.

Background. To evaluate the optical quality and related factors in patients with ocular hypertension (OHT). Methods. This was a prospective case-control study. A total of 12 eyes with OHT and 20 control eyes underwent testing with Optical Quality Analysis System II (OQAS II) to evaluate the modulation transfer function cut off frequency (MTF cutoff), the Strehl 2D ratio (SR), objective scatter index (OSI), tear-film mean OSI (TFOSI), and the OQAS values (OV100%,OV20%, and OV9%). Results. The optical quality of patients with OHT declined, with lower MTF cutoff (OHT 36.86 ± 7.11 cpd , controls 48.50 ± 4.04 cpd, t = -4.60, P < 0.05), lower SR (OHT 0.22 ± 0.04, controls 0.27 ± 0.05, t = -2.72, P < 0.05), lower OV100% (OHT 1.26 ± 0.25, controls 1.61 ± 0.14, t = -4.03, P < 0.05), lower OV20% (OHT 1.27 ± 0.27, controls 1.72 ± 0.20, t = -4.00, P < 0.05), and lower OV9% (OHT 1.30 ± 0.25, controls 1.69 ± 0.32, t = -2.28, P < 0.05). There were not any statistically significant differences in OSI and TFOSI. The MTF cutoff in patients with OHT was correlated significantly with age (r = -0.59, P < 0.05). Conclusions. Optical quality of patients with OHT is reduced, with lower MTF cutoff, SR, OV100%, OV20%, and OV9%. MTF cutoff is negatively related to age.

Assessment of visual outcomes of cataract surgery in Tujia nationality in Xianfeng County, China.

AIM: To evaluate the visual outcome and factors influencing visual outcome of manual small incision cataract surgery (MSICS) in the rural area in the Xianfeng County. METHODS: Eighty-two eyes of 82 patients who underwent cataract surgery performed by using MSICS technique were identified. Data collected included each patient's age, gender, the level of education. Uncorrected and corrected distance visual acuity (UDVA and CDVA) at presentation and at 1, 6, 8wk postoperatively, pre-existing eye disease, operative findings and complications, the risk factors were evaluated. RESULTS: In 82 patients, the average age was 69.6±0.6y, illiterate were 52 (63.4%). Of 82 eyes, pseudophakia was present in 77 eyes (93.9%). At 1wk postoperatively, 47 eyes (57.3%) had the UDVA of ≥6/18, and 52 eyes (63.4%) had the CDVA of ≥6/18. At 6 to 8wk postoperatively, 50 eyes (61.0%) had UDVA of ≥6/18, and 57 eyes (69.5%) had the CDVA of ≥6/18. Postoperative visual status was significantly related to the co-morbidities, such as corneal pathology, glaucoma (P<0.001). Operative complications, such as posterior capsule opacity and cystoid macular edema were main operative cause for the poor visual outcome. CONCLUSION: MSICS provides a good visual recovery in our study but the vision outcome did not fulfill the standards proposed by WHO, which highlights the need for an improvement in local socioeconomic understanding, population education and surgery quality.

Risk Factors for Dry Eye Syndrome: A Retrospective Case-Control Study.

PURPOSE: To investigate the independent risk factors of dry eye syndrome (DES) in Chinese. METHODS: A hospital-based age- and sex-matched population was enrolled with a case-control ratio of 1:2, with 789 DES case patients and 1119 healthy family members. Both groups underwent standard ophthalmologic examinations, including slit-lamp evaluation of the anterior segment, measurement of tear film breakup time, Schirmer test, and corneal fluorescein staining. Data on demographic characteristics and lifestyle habits were collected using a questionnaire. Dry eye syndrome risk factors were identified by univariate and multivariate logistic regression analyses. RESULTS: The following independent risk factors showed significant association with DES: diabetes (odds ratio [OR], 1.408; 95% confidence interval [CI], 1.031 to 1.924), hepatitis C (OR, 3.326; 95% CI, 1.632 to 6.776); connective tissue disease (OR, 2.157; 95% CI, 1.679 to 2.771), benign prostatic hyperplasia (OR, 3.892; 95% CI, 2.476 to 6.116), rosacea (OR, 3.747; 95% CI, 1.972 to 7.120), posttraumatic stress disorder (OR, 1.449; 95% CI, 1.043 to 2.013), hematopoietic stem cell transplantation (OR, 7.269; 95% CI, 2.312 to 22.849), head and neck radiotherapy (OR, 8.776; 95% CI, 3.096 to 24.873), postmenopausal estrogen therapy (OR, 1.912; 95% CI, 1.160 to 3.151), antihistamines (OR, 2.040; 95% CI, 1.516 to 2.746), antidepressants (OR, 1.982; 95% CI, 1.077 to 3.647), contact lenses (OR, 2.366; 95% CI, 1.266 to 4.423), and video display terminal exposure for more than 6 h/d (OR, 2.275; 95% CI, 1.451 to 3.568). Potentially protective factors against DES were vitamin supplements (OR, 0.716; 95% CI, 0.528 to 0.972) and Ω-3 fatty acid-rich diet (OR, 0.514; 95% CI, 0.332 to 0.796). CONCLUSION: Several known risk factors of DES are applicable to Chinese, and some distinctive dietary factors may be protective in this population.

Paxillin regulates vascular endothelial growth factor A-induced in vitro angiogenesis of human umbilical vein endothelial cells.

The purpose of the present study was to investigate the role of paxillin in the vascular endothelial growth factor A (VEGF­A)­induced adhesion, proliferation, migration and capillary formation of endothelial cells (ECs) in vitro. Human umbilical vein ECs (HUVECs) were used to evaluate these four processes in vitro. The HUVECs were either mock­transfected (control), transfected with scramble small interference RNA (siRNA) or transfected with siRNA specifically targeting paxillin. VEGF­A (20 ng/ml) was used to stimulate angiogenesis. The VEGF­A treatment significantly increased the adhesion, proliferation, migration and tube formation of the HUVECs in the control and scramble siRNA groups, whereas the siRNA­-mediated knockdown of paxillin inhibited these VEGF­A­induced effects. Paxillin is essential for VEGF­A­mediated angiogenesis in ECs and its inhibition may be a potential target for antiangiogenic therapies.

TNF-α stimulates MMP-2 and MMP-9 activities in human corneal epithelial cells via the activation of FAK/ERK signaling.

AIMS: Herpes simplex virus type-1-induced herpes simplex keratitis (HSK) is a common immunological cornea disease. While previous studies have addressed the role of tumor necrosis factor (TNF)-α and matrix metalloproteinases (MMPs) in HSK, the mechanistic link between TNF-α and MMPs in the pathogenesis of HSK remains elusive. METHODS: We first established a HSK mice model and measured the levels of TNF-α, MMP-2 and MMP-9 in the corneas at different time points by ELISA. Next, we employed cultured human corneal epithelial (HCE) cells as an in vitro model and performed gelatin zymography analysis. RESULTS: We observed that the change in the TNF-α level shared a similar pattern to that of MMP-2 and MMP-9 in the HSK mice model. Furthermore, TNF-α stimulated MMP-2 and MMP-9 activities in a dose-dependent manner, but either knockdown of focal adhesion kinase (FAK) by short interference RNA or inhibition of extracellular regulated protein kinase (ERK) by chemical inhibitor could block TNF-α-stimulated MMP-2 and MMP-9 activities in vitro. Taken together, our results provide in vivo evidence that the TNF-α level is positively correlated with MMP-2 and MMP-9 levels in a HSK model and in vitro evidence that TNF-α stimulates MMP-2 and MMP-9 activities via the activation of FAK/ERK signaling in HCE cells. CONCLUSIONS: Our findings shed new light on the pathogenesis of HSK and open up new possibility of modulating the TNF-α-FAK-ERK signaling cascade to pursue therapeutic measures for HSK.

In silico classification of human maximum recommended daily dose based on modified random forest and substructure fingerprint.

A modified random forest (RF) algorithm, as a novel machine learning technique, was developed to estimate the maximum recommended daily dose (MRDD) of a large and diverse pharmaceutical dataset for phase I human trials using substructure fingerprint descriptors calculated from simple molecular structure alone. This type of novel molecular descriptors encodes molecular structure in a series of binary bits that represent the presence or absence of particular substructures in the molecule and thereby can accurately and directly depict a series of local information hidden in this molecule. Two model validation approaches, 5-fold cross-validation and an independent validation set, were used for assessing the prediction capability of our models. The results obtained in this study indicate that the modified RF gave prediction accuracy of 80.45%, sensitivity of 75.08%, specificity of 84.85% for 5-fold cross-validation, and prediction accuracy of 80.5%, sensitivity of 76.47%, specificity of 83.48% for independent validation set, respectively, which are as a whole better than those by the original RF. At the same time, the important substructure fingerprints, recognized by the RF technique, gave some insights into the structure features related to toxicity of pharmaceuticals. This could help provide intuitive understanding for medicinal chemists.

[Effects of focal adhesional kinase signaling on TNF-α induced gelatinases activity in cornea epithelium].

OBJECTIVE: To investigate the effect of focal adhesional kinase (FAK) on tumor necrosis factor α (TNF-α)-induced MMP-2 and -9 activities in cornea epithelium. METHODS: Experimental research. The human corneal epithelial cells (HCE) were cultured in vitro. HCEs were incubated with different concentrations of TNF-α for 24 h, including 1 µg/L (group B), 10 µg/L (group C) and 100 µg/L (group D). The control group (group A) was incubated with phosphate buffer solution. The activities of MMPs were examined by gelatin zymography and the phosphorylation of FAK was examined by western blot analysis. FAK was down regulated by FAK siRNA following lipofectamine-mediated transfection in corneal epithelial cells. Down-regulation was confirmed using western blot analysis. Cells cultured with different concentrations of TNF-α (Groups B to D) and the control group (group A) was at similar volumes of media. Then the activities of MMP-2 and -9 were examined by gelatin zymography and the phosphorylation of FAK by western blot analysis. Statistical methods adopted one-way ANOVA and Tukey's honestly significant test between each group. RESULTS: Gelatin zymography: Activities of MMP-2 and -9 in TNF-α treated groups were greater than those of the control group. The activity of MMP-2 in A, B, C and D groups was 124.06 ± 4.06, 146.72 ± 5.51, 241.18 ± 5.65 and 389.95 ± 4.44, respectively with F = 2960.91, P = 0.000. The activity of MMP-9 in A, B, C and D groups was 122.78 ± 5.86, 165.70 ± 7.90, 479.49 ± 6.22 and 495.88 ± 5.03 (F = 4937.46, P = 0.000). Significant differences were found in each two groups (P = 0.000). Western blot analysis:the phosphorylation of FAK (p-FAK) in test groups (10-100 ng/ml) were significantly greater than that in control group (p-FAK of group C and D was 0.52 ± 0.03 and 0.61 ± 0.06, F = 431.03, P = 0.000). p-FAK levels in 100 ng/ml group were greater than that in 10 ng/ml group (P = 0.005). After down-regulating the protein FAK, TNF-α had no effect on the activity of MMP-2 (The data of MMP-2 were 55.13 ± 0.66, 55.67 ± 0.43, 55.49 ± 0.20 and 55.91 ± 0.37 in groups A, B, C and D, F = 2.73, P = 0.079). We detected the increasing activity of MMP-9 in group C, D and p-FAK in group D (The data of MMP-9's activity were 80.48 ± 0.39, 81.26 ± 0.62, 84.43 ± 0.47, 85.56 ± 0.61 in groups A, B, C and D, F = 105.80, P = 0.000). The activity of MMP-9 in group D was stronger than that from the group C (P = 0.019). We just only detected a small quantity of p-FAK in group D (0.47 ± 0.05), which was weaker than that before down regulating the protein FAK (t = 5.03, P = 0.001). CONCLUSION: Our results demonstrate the critical role of FAK in TNF-α induced activity of MMP-2 and -9 in human corneal epithelium cells. Blocking the FAK signaling pathway can reduce the activity of MMP-2 and -9 which may play an important role in prevention and treatment of corneal diseases.

[Differentiation and apoptosis effects of all-trans retinoic acid on inducing umbilical cord mesenchymal stem cells into neuron-like cells].

OBJECTIVE: To evaluate the role of different concentration of all-trans retinoic acid (ATRA) on the morphology, proliferation and apoptosis in inducing umbilical cord mesenchymal stem cells (MSC) into neuron-like cells in vitro, and screen the optimal concentration of ATRA. METHODS: It was an experimental study. The third passage of MSC was placed in 24-well cell culture plates at a density of 1 × 10(4)/well. After the adherent of cells, the medium was changed to DMEM/F-12 containing different concentration of ATRA (0.25 µmol/L, 0.5 µmol/L, 1.0 µmol/L, 2.0 µmol/L, 4.0 µmol/L) for 24 h respectively. The cells cultured without ATRA were taken as the control group. After another 24 h, the morphologic changes of induced cells were observed by inverted microscope and cell proliferation, apoptosis of ATRA was analyzed using the MTT colorimetric assay. We take another control group and ATRA groups to detect the apoptotic and positive stained percentage of induced cells by Annexin V-FITC/PI combining flow cytometry. The optimal concentration of ATRA was determined by all the above-mentioned index. According to the nature of the material, analysis of variance (ANOVA) was employed for absorption value and apoptosis rate in different concentration of ATRA for 24 h, t test for further comparison between two groups. T-test were also used between the positive expression of induced neuron-like cells and the control group. RESULTS: Compared to the control group, ATRA at the concentration of 0.25 µmol/L did not inhibit the proliferation of umbilical cord MSC obviously (t = 0.72, 1.32, P > 0.05). Part of MSC were floating instantly at the moment of adding ATRA of 4.0 µmol/L and no adherent cells were observed after 24 h' culture. Exposed to ATRA at the concentration of ≥ 1.0 µmol/L for 24 h, the proliferation of MSC were significantly inhibited, showing a dose-dependent manner (t = 8.8, 18.9, 22.1; P < 0.01). 0.5 µmol/L of ATRA did not affect the proliferation of cells and its morphology remained normal; 1.0 µmol/L of ATRA affected very few cells; but 2.0 µmol/L of ATRA cultured for 24 h inhibited the proliferation of cells obviously than 1 h, and the cells increased in size and became flattened. Flow cytometry showed that the rate of apoptosis between the control group and ≥ 1.0 µmol/L groups were significantly different (t = 9.88, 19.95, 31.61; P < 0.01). CONCLUSION: In the process of inducing umbilical cord MSC into neuron-like cells, 0.5 µmol/L ATRA was the optical concentration. ≥ 1.0 µmol/L ATRA can inhibit the cell proliferation, increase the apoptosis of cells significantly and caused obvious damages.

[Expression of matrix metalloproteinases in experimental herpes simplex virus keratitis].

OBJECTIVE: To determine the distribution and activities of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) during the course of experimental herpes simplex virus type-1 (HSV-1) keratitis. METHODS: Keratitis was induced in BALB/c mice by inoculating the cornea with 10(5) plaque-forming units (pfu) of HSV-1 (KOS strain). Corneas were harvested at days 0, 2, 7, 14 and 28 post-infection. Expression of MMP-2, MMP-9, MMP-8, TIMP-1 and TIMP-2 were detected by immunohistochemistry and Western blot. The enzymatic activities were analyzed by Zymography. RESULTS: At day 2 post-infection, MMP-2 and MMP-9 expression were increased in the epithelium as compared to the uninfected control eyes, and were detected in the superficial stroma and in inflammatory cells beneath the epithelium. Similar staining patterns were detected for TIMP-1 and TIMP-2. MMP-2 and MMP-9 epithelial staining persisted until day 28 post-infection. Necrotizing keratitis with corneal ulceration was present on days 14 and 28 post-infection. This correlated with increased expression of MMP-2 and MMP-9 within the stroma and in infiltrating inflammatory cells. MMP-2, MMP-9, TIMP-1 and TIMP-2 staining were particularly intense in the proximity of the ulcers. The neutrophils, which were abundant at the site of ulceration, were stained positive with MMP-8. Both gelatinolytic activities and caseinolytic activities were upregulated after HSV-1 corneal infection. CONCLUSIONS: Our data suggest that MMPs produced by resident corneal cells and by inflammatory cells possibly promote epithelial keratitis and ulcerative process after corneal HSV-1 infection. The interaction of MMPs and TIMPs may regulate the course of necrotizing HSV keratitis.

Increased expression of nuclear factor-kappaB/RelA is correlated with tumor angiogenesis in human colorectal cancer.

BACKGROUND AND AIMS: Recent studies have shown that nuclear factor-kappa B/RelA (NF-kappa B/RelA) is involved in tumor angiogenesis. This study examined whether NF-kappa B/RelA expression is associated with vascular endothelial growth factor (VEGF) expression and microvessel density in human colorectal cancer. MATERIALS AND METHODS: Ten specimens from normal colorectal mucosa and 52 colorectal adenocarcinomas were obtained by surgery or endoscopy. Immunohistochemical expression of NF-kappa B/RelA, VEGF, and CD34 was detected on paraffin-embedded tissue sections. RESULTS: NF-kappa B/RelA and VEGF were significantly overexpressed and associated with microvessel density in colorectal cancer. A significant association was found between NF-kappa B/RelA and VEGF expression. Clinicopathological features were not correlated with NF-kappa B/RelA, VEGF expression, or microvessel density. CONCLUSION: Our results suggest that increased expression of NF-kappa B/RelA contributes to tumor angiogenesis in colorectal cancer. VEGF may play an important role in mediating the NF-kappa B/RelA angiogenic pathway.

Matrix metalloproteinases (MMP-2 and 9) and tissue inhibitors of matrix metalloproteinases (TIMP-1 and 2) during the course of experimental necrotizing herpetic keratitis.

To determine the distribution and activities of metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) during the course of experimental herpes simplex virus (HSV) type-1 keratitis, BALB/c mice were corneally infected with 10(5) plaque-forming units (PFU) of HSV-1 (KOS strain) and then observed for the clinical signs of keratitis. Corneas were harvested at days 0, 2, 7 and 14 post-infection (p.i.). MMP-2, MMP-9, MMP-8, TIMP-1 and TIMP-2 were detected by immunohistochemistry and the Western blot technique. The enzymatic activities were analyzed by zymography. Epithelial HSV keratitis was present at day 2 after corneal infection and healed by day 5 p.i. While the expression and activity of MMP-2, MMP-8 and MMP-9 increased in the corneas at day 2 p.i., it was reduced at day 7 p.i. TIMP-1 and -2 were expressed in the corneas before and seven days after infection. Necrotizing stromal keratitis with corneal ulceration and dense polymorphonuclear leukocyte (PMN) infiltration was present at day 14 p.i. This correlated with increased expression of MMP-2, MMP-8 and MMP-9 in the corneas. MMP-8, MMP-9 and MMP-2 staining was particularly intense in the proximity of the ulcers and in areas of PMN infiltration. At day 14 p.i., MMP-2, -8 and -9 activities were upregulated, and TIMP-2 was expressed. These data suggest that MMPs produced by resident corneal cells and PMNs may possibly play a role in early epithelial keratitis and in the ulcerative process in the late phase after corneal HSV-1 infection. The ratio of MMPs to TIMPs may be important for the course of necrotizing HSV keratitis. TIMPs might participate in the repair process.

Inhibition of cytosolic phospholipase A2 mRNA expression: a novel mechanism for acetylsalicylic acid-mediated growth inhibition and apoptosis in colon cancer cells.

Acetylsalicylic acid (ASA) has been confirmed to inhibit proliferation and to induce apoptosis in human colorectal cancer cells in vitro. However, the mechanism by which ASA exhibits antiproliferative and proapoptotic effects in cyclooxygenase 2 (COX-2)-negative cells remains to be further elucidated. In the present study, SW480, a COX-2-negative colon cancer cell line, was treated with various concentrations of ASA (0, 2.5, 5, and 10 mM). The antiproliferative and proapoptotic effects of ASA were confirmed by MTT assay, flow cytometry of propidium iodide (PI)-stained cells, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. After treatment with ASA, intracellular cyclic AMP (cAMP) levels were increased and the production of prostaglandin E2 (PGE2) was decreased. RT-PCR analysis revealed that treatment of ASA induced a concentration-dependent downregulation of cytosolic phospholipase A2 (cPLA2) mRNA expression in SW480 cells and also in two other colorectal cancer cell lines, Colo320 and HT-29 cells. Intracellular calcium levels were unaffected by ASA treatment. Our results indicate that the ASA-induced downregulation of cytosolic phospholipase A2 mRNA expression might be a novel mechanism for ASA-mediated growth inhibition and apoptosis in colon cancer cells.