Comparison of immunochemotherapy and chemotherapy alone in conversion therapy for locally advanced unresectable esophageal squamous cell carcinoma.

Background: The clinical value of preoperative immunochemotherapy and simple chemotherapy induction regimen in the conversion therapy of locally advanced unresectable esophageal squamous cell carcinoma (ESCC) is still unclear. Method: Retrospective analysis was conducted on patients with unresectable cT4b stage ESCC who underwent conversion surgery in our hospital from January 2020 to December 2022. According to the preoperative induction treatment plan, they were divided into induction immunochemotherapy group (iICT group) and induction chemotherapy group (iCT group). The conversion surgery rate, R0 resection rate, radiological and pathological tumor responses, safety, and short-term survival outcomes were analyzed. Results: The results showed that a total of 199 patients with cT4b locally advanced unresectable ESCC who underwent preoperative induction therapy were included in this study. Among them, there were 64 cases (32.2%) in the iICT group, 135 cases (67.8%) in the iCT group. There was a statistically significant difference in objective response rate (73.5% vs 48.9%) and conversion surgery rate (81.3% vs 66.7%), between the iICT and iCT groups (P=0.001 and P=0.019). Among the two groups of patients who underwent surgery, there were statistically significant differences in R0 resection rate (94.2% vs 82.2%) and pathological complete remission rate (23.1% vs 6.7%) between the iICT and iCT groups (P=0.043 and P=0.004). And there was no statistically significant difference in the incidence of grade 3 and above between two groups (P=0.928). The 2-year EFS of the iICT group and iCT group were 76.4% and 42.4%, respectively, with statistically significant differences (P=0.006). Conclusions: Compared with simple chemotherapy, the combination of PD-1 inhibitors and chemotherapy can achieve better conversion surgery rate, tumor response and event-free survival in the conversion therapy of locally advanced unresectable ESCC.

Psoas muscle mass index and peak expiratory flow as measures of sarcopenia: relation to outcomes of elderly patients with resectable esophageal cancer.

Objectives: The objective of this study is to investigate whether the evaluation of postoperative outcomes or overall survival in patients who undergo surgery for esophageal cancer can be achieved by assessing sarcopenia using psoas muscle mass index and peak expiratory flow. Methods: This retrospective study analyzed the clinical data of 356 elderly patients (≥ 65 years) who had undergone radical surgery for esophageal cancer. Muscle mass and muscle strength were assessed by psoas muscle mass index (bilateral psoas area/height2) and peak expiratory flow, using preoperative computed tomography and spirometry, respectively. Sarcopenia is defined as a condition where both the psoas muscle mass index and peak expiratory flow fall below their gender-specific cutoff values. Survival and postoperative complications were compared between patients with and without sarcopenia. Results: Out of the 356 elderly individuals diagnosed with esophageal cancer, 84 patients (23.6%) were found to have sarcopenia. The group with sarcopenia showed a notably higher occurrence of postoperative pneumonia (29.8% vs 16.9%, P < 0.001) and anastomotic leak (9.5% vs 3.7%, P < 0.05) compared to those without sarcopenia. Additionally, a multivariate analysis concluded that sarcopenia independently acted as a risk factor for postoperative pneumonia, possessing an odds ratio of 1.90 (P < 0.05). The survival rate after 3 years for individuals with sarcopenia was considerably lower than those without sarcopenia (57.8% vs 70.2%, P < 0.05). Sarcopenia was identified as an unfavorable prognostic factor for overall survival, with a hazard ratio of 1.51 (P < 0.05). Conclusions: Preoperative sarcopenia diagnosed by psoas muscle mass index and peak expiratory flow is associated with reduced overall survival and adverse postoperative outcomes among elderly individuals suffering from esophageal cancer.

Warning and Nursing Experience of Anesthesia Depth Monitoring for Patients with General Anesthesia Delayed to Leave Anesthesia Recovery Room and Delirium.

Affected by the residues of narcotic drugs, patients under general anesthesia are vulnerable to emergence of agitation, delirium, hemodynamic changes, and other adverse events in the recovery period of anesthesia. Therefore, it is necessary to strengthen the observation and care of these patients. Depth of anesthesia monitoring (DAM) has always been a concern for anesthesiologists, but there are few reports related to it. This study compared the early warning value of DAM for patients under general anesthesia with delayed exit from the anesthesia recovery unit (PACU) and delirium and summarized the related nursing experience. The results showed that DAM could reduce the incidence of complications in patients under general anesthesia, reduce the incidence of delirium, shorten the time of postoperative anesthesia recovery and PACU observation time, reduce the workload of nursing staff, and improve nursing satisfaction. DAM plays an important role in improving the quality and efficiency of care in PACU.

Self-assembled RNA nanocarrier-mediated chemotherapy combined with molecular targeting in the treatment of esophageal squamous cell carcinoma.

BACKGROUND: Esophageal cancer is the fifth most common cancer affecting men in China. The primary treatment options are surgery and traditional radio-chemotherapy; no effective targeted therapy exists yet. Self-assembled RNA nanocarriers are highly stable, easily functionally modified, and have weak off-tumor targeting effects. Thus, they are among the most preferred carriers for mediating the targeted delivery of anti-tumor drugs. miR-375 was found to be significantly down-regulated in esophageal squamous cell carcinoma (ESCC) tissues and its overexpression effectively inhibits the proliferation, migration, and invasion of ESCC cells. Moreover, epidermal growth factor receptor (EGFR) was overexpressed in ESCC cells, and accumulation of RNA nanoparticles in ESCC tumors was enhanced by EGFR-specific aptamer (EGFRapt) modification. RESULTS: Herein, a novel four-way junction RNA nanocarrier, 4WJ-EGFRapt-miR-375-PTX simultaneously loaded with miR-375, PTX and decorated with EGFRapt, was developed. In vitro analysis demonstrated that 4WJ-EGFRapt-miR-375-PTX possesses strong thermal and pH stabilities. EGFRapt decoration facilitated tumor cell endocytosis and promoted deep penetration into 3D-ESCC spheroids. Xenograft mouse model for ESCC confirmed that 4WJ-EGFRapt-miR-375-PTX was selectively distributed in tumor sites via EGFRapt-mediating active targeting and targeted co-delivery of miR-375 and PTX exhibited more effective therapeutic efficacy with low systemic toxicity. CONCLUSION: This strategy may provide a practical approach for targeted therapy of ESCC.

Pigment Epithelium-Derived Factor Promotes the Growth and Migration of Human Esophageal Squamous Cell Carcinoma.

Pigment epithelium-derived factor (PEDF) is an oncogene found in various types of cancers. However, how PEDF affects the development of human esophageal squamous cell carcinoma (ESCC) is unknown. This study investigates the role of PEDF in ESCC cell proliferation, migration, and cell cycle both in vitro and in vivo. The PEDF expression was examined in patient tumor samples and ESCC cell lines. Short hairpin RNA technology was used to inhibit the PEDF expression in ESCC EC9706 and KYSE150 cells. In vitro cell proliferation and migration assays were performed. The effects of PEDF on tumor growth and progression were examined in vivo in murine subcutaneous xenograft tumor models. It was found that PEDF was overexpressed in esophageal cancer cells and patient tumor tissues compared to normal control samples. PEDF enhanced cell cycle progression and inhibited cell apoptosis. Knock down of PEDF inhibited esophageal cell proliferation and migration in vitro. Moreover, Inhibition of PEDF significantly reduced tumor growth and tumor size in vivo. These results indicate that PEDF induce tumorigenesis in ESCC and can be a potential therapeutic target for cancer treatment.

Dysregulated circRNAs and ceRNA network in esophageal squamous cell carcinoma.

Accumulating evidence suggests that circular RNA (circRNA), once thought to be a transcriptional error, plays an important regulatory role in the tumor biological process. Some circRNAs regulate the protein-coding gene expression by competitive binding with microRNAs (miRNAs). However, functional roles of circRNA-mediated competitive endogenous RNAs (ceRNAs) in esophageal squamous cell carcinoma (ESCC) are rarely reported. To explore the biological functions of circRNAs in ESCC, we surveyed the integrating differential circRNA expression of ESCC and para-cancer tissues using microarray in three patients. Then, we screened out differentially expressed mRNAs obtained from 81 ESCC tissues and 11 normal tissues in The Cancer Genome Atlas (TCGA). Then, we constructed a hypothetical ceRNA network by integrating differential expression of circRNAs and mRNAs. Finally, 32 differentially expressed circRNAs and 98 differentially expressed mRNAs were linked by 64 miRNAs to build the ceRNA network in ESCC. We suggest that the identified ceRNA network can facilitate a better understanding of circRNA-related mechanisms in ESCC.

Long noncoding RNA AFAP1-AS1 is upregulated in NSCLC and associated with lymph node metastasis and poor prognosis.

Long noncoding RNA (lncRNA) has been indicated to have an important role in various types of malignant tumors; however, only a small number of lncRNAs have been entirely elucidated. In the present study, a novel lncRNA, actin filament associated protein 1 antisense RNA 1 (AFAP1-AS1), was investigated, which is highly expressed in non-small cell lung cancer (NSCLC). Reverse transcription-quantitative polymerase chain reaction and in situ hybridization were performed to detect AFAP1-AS1 expression in frozen tissues and tissue microarrays, respectively. The results revealed that the expression level of AFAP1-AS1 was significantly increased in tumor tissues, compared with the paired non-cancerous tissues. It was also determined that the AFAP1-AS1 expression level was higher in patients with lymph node metastasis than those without lymph node metastasis (P=0.014). Kaplan-Meier analysis was conducted to evaluate the overall survival of patients with NSCLC and different expression levels of AFAP1-AS1, and the results indicated that patients with high AFAP1-AS1 expression had a reduced survival time, compared with those with low AFAP1-AS1 expression (P=0.011). Cox regression analysis was also performed to analyze the prognostic value of lncRNA AFAP1-AS1. The obtained data demonstrated that lncRNA AFAP1-AS1 was an unfavorable prognostic biomarker for NSCLC (HR: 3.12, 95% CI (1.05-9.25), P=0.040). In conclusion, it was demonstrated that lncRNA AFAP1-AS1 is overexpressed in NSCLC and an unfavorable biomarker for patients with NSCLC.

Choice of postoperative radiation for stage IIIA pathologic N2 non-small cell lung cancer: impact of metastatic lymph node number.

BACKGROUND: Postoperative radiation (PORT) is an option for non-small cell lung cancer (NSCLC) patients with resectable stage IIIA pathological N2 status (pN2). For patients with PORT, this study aims to investigate the impact of the exact number of positive lymph nodes (LNs) on overall survival (OS) and lung cancer-specific survival (LCSS). METHODS: Within the Surveillance, Epidemiology, and End Results database, we identified 3373 patients with stage IIIA pathological N2 status (pN2) NSCLC who underwent a lobectomy or pneumonectomy from 2004 to 2013. OS and LCSS were compared among patients coded as receiving PORT or observation. The proportional hazards model was applied for investigation. RESULTS: OS and LCSS favored PORT for patients with stage IIIA (pN2) NSCLC. Multivariable analyses showed that PORT and the exact number of positive LNs (n ≤ 3) were independently associated with better OS and LCSS. Both better OS and LCSS emerged for positive LNs (n > 3) after the use of PORT in survival analyses, whereas the benefits of OS and LCSS were not observed anymore for positive LNs (n ≤ 3) group. More importantly, multivariable analyses showed that the use of PORT is an independent risk factor of survival for positive LNs (n > 3) but not for positive LNs (n ≤ 3). CONCLUSIONS: In Stage IIIA (pN2) NSCLC, the use of PORT demonstrated better survival results than no PORT for patients with positive LNs (n > 3), but not for patients with positive LNs (n ≤ 3).

[Application of bundles of intervention in the treatment of esophageal carcinoma anastomotic leak].

OBJECTIVE: To investigate the application of bundles of intervention in the treatment of esophageal carcinoma anastomotic leak. METHODS: From January 2014 to May 2015, 44 cases of esophageal carcinoma anastomotic fistula were treated by bundles of intervention (through the collection of a series of evidence-based treatment and care measures for the treatment of diseases) in Department of Thoracic Surgery, Huai'an First Hospital, Nanjing Medical University (bundles of intervention group), and 68 patients with esophageal carcinoma postoperative anastomotic leak from December 2013 to January 2012 receiving traditional therapy were selected as the control group. The clinical and nutritional indexes of both groups were compared. RESULTS: There were no significant differences in general data and proportion of anastomotic leak between the two groups. Eleven patients died during hospital stay, including 3 cases in bundles of intervention group(6.8%) and 8 cases in control group (11.8%) without significant difference(P = 0.390). In bundles of intervention group, 1 case died of type III( intrathoracic anastomotic leak, 2 died of type IIII( intrathoracic anastomotic leak. In control group, 2 cases died of type III( cervical anastomotic leak, 2 died of type III( intrathoracic anastomotic leak and 4 of type IIII( intrathoracic anastomotic leak. The mortality of bundles of intervention group was lower than that of control group. The duration of moderate fever [(4.1±2.4) days vs. (8.3±4.4) days, t=6.171, P=0.001], the time of antibiotic use [(8.2±3.8) days vs.(12.8±5.2) days, t=5.134, P = 0.001], the healing time [(21.5±12.7) days vs.(32.2±15.8) days, t=3.610, P=0.001] were shorter, and the average hospitalization expenses[(63±12) thousand yuan vs. (74±19) thansand yuan, t=3.564, P=0.001] was lower in bundles of intervention group than those in control group. Forty-eight hours after occurrence of anastomotic leak, the levels of hemoglobin, albumin and prealbumin were similar in both groups. However, at the time of fistula healing, the levels of hemoglobin [(110.6±10.5) g/L vs.(103.8±11.1) g/L, t=3.090, P=0.002], albumin [(39.2±5.2) g/L vs.(36.3±5.9) g/L, t=2.543, P=0.013] and prealbumin [(129.3±61.9) g/L vs.(94.1±66.4) g/L, t=2.688, P=0.008] were significantly higher in bundles of intervention group. CONCLUSION: In the treatment of postoperative esophageal carcinoma anastomotic leak, application of bundles of intervention concept can significantly improve the nutritional status and improve the clinical outcomes.

High expression of long non-coding RNA SBF2-AS1 promotes proliferation in non-small cell lung cancer.

BACKGROUND: Recent evidence has proven that long noncoding RNAs (lncRNAs) play important roles in cancer biology, while few lncRNAs have been characterized in NSCLC. Here, we characterized a novel lncRNA, SBF2 antisense RNA 1 (SBF2-AS1), in non-small cell lung cancer (NSCLC). METHODS: Quantitative real-time PCR was used to quantify SBF2-AS1 expression in NSCLC tissues and cell lines. The correlation of SBF2-AS1 expression with clinicopathologic features was analyzed in a cohort NSCLC patient. Loss of function and gain of function studies were performed to determine the effects of SBF2-AS1 on proliferation and metastasis of NSCLC cells. RNA immunoprecipitation and chromosome immunoprecipitation assay was performed to confirm the interaction between SBF2-AS1 with protein and chromosome. RESULTS: We confirmed that SBF2-AS1 was significantly upregulated in NSCLC compared with corresponding non-tumor tissues, and a high expression level of SBF2-AS1 was correlated with lymph node metastasis and advanced TNM stage. Using siRNAs specifically targeting SBF2-AS1 and plasmid vector, we successfully silenced and overexpressed SBF2-AS1 in NSCCLC cell lines and investigated its biological function both in vitro and in vivo. After the silencing of SBF2-AS1, the metastasis of NSCLC cells was significantly inhibited, the silencing of SBF2-AS1 decreased the proliferation of NSCLC cells, and the cell cycle was arrested at the G1 phase; while overexpression promoted proliferation ability. Xenograft tumor models revealed that the silencing of SBF2-AS1 inhibited tumor growth in vivo. We speculated that SBF2-AS1 might negatively regulate P21. RNA immunoprecipitation discovered that SBF2-AS2 could bind with a core component of polycomb repressive complex2, SUZ12. Additionally chromatin immunoprecipitation assay demonstrated that, after silencing SBF2-AS1, the enrichment of SUZ12 and trimethylation of histone 3 lysine 27 decreased at the promoter region of P21. CONCLUSIONS: We demonstrated that SBF2-AS1 is upregulated in NSCLC and promotes proliferation of NSCLC tumor cells. SBF2-AS1 may serve as a novel biomarker and potential therapeutic target for NSCLC patients.

Relationship of Fas, FasL, p53 and bcl-2 expression in human non-small cell lung carcinomas.

OBJECTIVE: Lack of surface Fas expression is a main route for apoptotic resistance which is considered an important mechanism of tumorigenesis and tumor progression. Fas and FasL expression in 110 non-small cell lung carcinomas (NSCLCs) were investigated to evaluate their roles in pulmonary carcinogenesis and to examine the clinicopathologic significance of Fas expression with its relationship with p53 and bcl-2 over- expression. METHODS: Immunohistochemical analysis using tissue microarray demonstrated that a large proportion of NSCLC patients (60%) showed lack of membranous Fas expression. The Fas-negative cases revealed the significantly lower survival rate than Fas-positive ones. Also, the loss of Fas receptor expression was found more frequently in advanced stage and higher nodal status. FasL protein was increased in most NSCLCs (89%) compared to normal lungs. RESULTS: p53 and bcl-2 overexpression showed no association with Fas expression. Conclusively, reduced membranous Fas expression as a mechanism of apoptotic resistance is considered to play an important part of the pulmonary carcinogenesis, which may predict poor survival and have a negative prognostic influence. CONCLUSION: Increased FasL expression is thought to be a basis for the immune evasion in NSCLCs. The rare bcl-2 overexpression suggests that this anti-apoptotic protein is unlikely to play a role in the apoptotic resistance of NSCLCs.

GlideScope-assisted fiberoptic bronchoscope intubation in a patient with severe rheumatoid arthritis.

Here, we report that, under the assistance of both the GlideScope and a fiberoptic bronchoscope, tracheal intubation was accomplished successfully in a 50-year-old woman with severe rheumatoid arthritis who underwent tongue lump resection under general anesthesia. Either the GlideScope or the fiberoptic bronchoscope alone failed to secure the airway; the use of both in combination facilitated airway intubation. This case report indicate that, even with careful preoperative assessment, patients who suffer from rheumatoid arthritis may have severe airway difficulty with intubation, and the combined use of the GlideScope and a fiberoptic bronchoscope can be a novel alternative for tracheal intubation in patients with severe airway difficulty.

Partial removal of the pulmonary artery in video-assisted thoracic surgery for non-small cell lung cancer.

Lobectomy with partial removal of the pulmonary artery in video-assisted thoracic surgery (VATS) currently remains a challenge for thoracic surgeons. We were interested in introducing pulmonary vessel blocking techniques in open thoracic surgery into video-assisted thoracic surgery (VATS) procedures. In this study, we reported a surgical technique simultaneously blocking the pulmonary artery and the pulmonary vein for partial removal of the pulmonary artery under VATS. Seven patients with non-small-cell lung cancer (NSCLC) received lobectomy with partial removal of the pulmonary artery using the technique between December 2007 and March 2012. Briefly, rather than using a small clamp on the distal pulmonary artery to the area of invading cancer, we replaced a vascular clamp with a ribbon and Hem-o-lock clip to block the preserved pulmonary veins so as to prevent back bleeding and yield a better view for surgeons. The mean occlusion time of the pulmonary artery and pulmonary veins were 44.0±10.0 and 41.3±9.7 minutes, respectively. The mean repair time of the pulmonary artery was 25.3±13.7 minutes. No complications occurred. No patients showed abnormal blood flow through the reconstructed vessel. There were no local recurrences on the pulmonary artery. In conclusion, the technique for blocking the pulmonary artery and veins is feasible and safe in VATS and reduces the risk of abrupt intraoperative bleeding and the chance of converting to open thoracotomy, and extends the indications of VATS lobectomy.

Ex vivo corneal epithelial wound healing following exposure to ophthalmic nonsteroidal anti-inflammatory drugs.

PURPOSE: Ketorolac 0.45% is a new formulation of topical ketorolac in which preservative (benzalkonium chloride, BAK) was removed and carboxymethylcellulose (CMC) was added to improve tolerability and reduce dosing frequency. This study compared the effects of ketorolac 0.45% on corneal wound healing to prior ketorolac formulations (0.4% and 0.5%), bromfenac 0.09%, and nepafenac 0.1%. METHODS: Two parallel-group comparisons were performed in series. A 5-mm central epithelial wound was made in fresh porcine corneas. After 24 hours in minimum essential medium (MEM), corneas were incubated for 10 minutes with study drugs, Triton X-100 1% (positive control), or MEM (negative control), followed by 24 hours in MEM. The remaining wound area was stained, photographed, and quantified (pixels). Study 1 compared ketorolac 0.45% to ketorolac 0.4% and ketorolac 0.5%. Study 2 compared ketorolac 0.45% to bromfenac 0.09% and nepafenac 0.1%. RESULTS: The mean (±SD) original wound area was 200,506 ± 4,363 pixels, which was reduced to 59,509 ± 4850 at 48 hours after exposure to Triton X-100 1%. In study 1, the mean remaining wound areas at 48 hours in pixels were 2969 ± 1633 with MEM, 586 ± 299 with ketorolac 0.45% (significantly reduced, P < 0.05 vs all other treatments), 10,228 ± 7541 with ketorolac 0.4%, and 50,674 ± 33,409 with ketorolac 0.5% (significantly enlarged, P < 0.05 vs MEM). In study 2, the mean remaining wound areas at 48 hours were 565 ± 1263 with MEM, 322 ± 229 with ketorolac 0.45% (significantly reduced, P < 0.01 vs bromfenac 0.09% and nepafenac 0.1%), 29,093 ± 14,295 with bromfenac 0.09% (significantly enlarged, P <0.01 vs MEM) and 47,322 ± 13,736 with nepafenac 0.1% (significantly enlarged, P < 0.01 vs MEM and vs bromfenac 0.09%). CONCLUSION: Corneas treated with ketorolac 0.45% healed as rapidly as those treated with MEM, likely secondary to addition of CMC and removal of BAK. In the ex vivo corneal organ culture model, ketorolac 0.45% had statistically less impact on corneal re-epithelialization than prior ketorolac formulations (0.4% and 0.5%), bromfenac 0.09%, and nepafenac 0.01%.

Impaired epithelial wound healing and EGFR signaling pathways in the corneas of diabetic rats.

PURPOSE. The purpose of the study was to investigate the effects of hyperglycemia on EGFR (epidermal growth factor receptor)-mediated wound response and signal transduction in the corneal epithelium of rats with type I diabetes mellitus (DM). METHODS. Corneal epithelia were removed from streptozotocin (STZ)- and weight-matched normal rats. Wound healing was monitored by fluorescein staining at 24 or 48 hours after epithelial debridement. Phosphorylation of EGFR, AKT, ERK, and BAD was determined by Western blot analysis. The distribution of phospho-AKT and proliferating cell nuclear antigen (PCNA) in rat corneas was examined by immunohistochemistry. Cell death was evaluated by TUNEL staining. RESULTS. A significant delay in corneal epithelial wound healing was observed 48 hours after wounding in the diabetic rats compared with the weight-matched control rats. In the DM rat corneas, epithelial cells demonstrated diminished responses to wounding, as assessed by the phosphorylation of EGFR and its downstream signaling molecules, AKT and ERK. Furthermore, although the distribution pattern of phospho-AKT suggested a role for AKT in epithelial migration and proliferation in the normoglycemic rat corneas, it was abrogated in the healing epithelia of the DM rats. Consistent with impaired AKT activity, the number of PCNA-stained cells was also greatly reduced in the healing corneas of the diabetic rats. Finally, decreases in pBAD (Ser(136) and Ser(112)) and increases in TUNEL-positive cells were observed in both the uninjured and healing corneal epithelia of the DM rats, but not of the control rats. CONCLUSIONS. In the corneas of SZT rats, EGFR-PI3K-AKT and ERK, as well as their downstream BAD signaling pathways in migratory epithelium, were altered, resulting in increased apoptosis, decreased cell proliferation, and delayed wound closure.

UBIAD1 mutation alters a mitochondrial prenyltransferase to cause Schnyder corneal dystrophy.

BACKGROUND: Mutations in a novel gene, UBIAD1, were recently found to cause the autosomal dominant eye disease Schnyder corneal dystrophy (SCD). SCD is characterized by an abnormal deposition of cholesterol and phospholipids in the cornea resulting in progressive corneal opacification and visual loss. We characterized lesions in the UBIAD1 gene in new SCD families and examined protein homology, localization, and structure. METHODOLOGY/PRINCIPAL FINDINGS: We characterized five novel mutations in the UBIAD1 gene in ten SCD families, including a first SCD family of Native American ethnicity. Examination of protein homology revealed that SCD altered amino acids which were highly conserved across species. Cell lines were established from patients including keratocytes obtained after corneal transplant surgery and lymphoblastoid cell lines from Epstein-Barr virus immortalized peripheral blood mononuclear cells. These were used to determine the subcellular localization of mutant and wild type protein, and to examine cholesterol metabolite ratios. Immunohistochemistry using antibodies specific for UBIAD1 protein in keratocytes revealed that both wild type and N102S protein were localized sub-cellularly to mitochondria. Analysis of cholesterol metabolites in patient cell line extracts showed no significant alteration in the presence of mutant protein indicating a potentially novel function of the UBIAD1 protein in cholesterol biochemistry. Molecular modeling was used to develop a model of human UBIAD1 protein in a membrane and revealed potentially critical roles for amino acids mutated in SCD. Potential primary and secondary substrate binding sites were identified and docking simulations indicated likely substrates including prenyl and phenolic molecules. CONCLUSIONS/SIGNIFICANCE: Accumulating evidence from the SCD familial mutation spectrum, protein homology across species, and molecular modeling suggest that protein function is likely down-regulated by SCD mutations. Mitochondrial UBIAD1 protein appears to have a highly conserved function that, at least in humans, is involved in cholesterol metabolism in a novel manner.

Growth factors and corneal epithelial wound healing.

In this article, we briefly review recent findings in the effects of growth factors including the EGF family, KGF, HGF, IGF, insulin, and TGF-beta on corneal epithelial wound healing. We discuss the essential role of EGFR in inter-receptor cross-talk in response to wounding in corneal epithelium and bring forward a concept of "alarmins" to the field of wound healing research.

High glucose suppresses epidermal growth factor receptor/phosphatidylinositol 3-kinase/Akt signaling pathway and attenuates corneal epithelial wound healing.

OBJECTIVE: Patients with diabetes are at an increased risk for developing corneal complications and delayed wound healing. This study investigated the effects of high glucose on epidermal growth factor receptor (EGFR) signaling and on epithelial wound healing in the cornea. RESEARCH DESIGN AND METHODS: Effects of high glucose on wound healing and on EGFR signaling were investigated in cultured porcine corneas, human corneal epithelial cells, and human corneas using Western blotting and immunofluorescence. Effects of high glucose on reactive oxygen species (ROS) and glutathione levels and on EGFR pathways were assessed in porcine and primary human corneal epithelial cells, respectively. The effects of EGFR ligands and antioxidants on high glucose-delayed epithelial wound healing were assessed in cultured porcine corneas. RESULTS: High glucose impaired ex vivo epithelial wound healing and disturbed cell responses and EGFR signaling to wounding. High glucose suppressed Akt phosphorylation in an ROS-sensitive manner and decreased intracellular glutathione in cultured porcine corneas. Exposure to high glucose for 24 h resulted in an increase in ROS-positive cells in primary human corneal epithelial cells. Whereas heparin-binding EGF-like growth factor and antioxidant N-acetylcysteine had beneficial effects on epithelial wound closure, their combination significantly accelerated high glucose-delayed wound healing to a level similar to that seen in control subjects. Finally, Akt signaling pathway was perturbed in the epithelia of human diabetic corneas, but not in the corneas of nondiabetic, age-matched donors. CONCLUSIONS: High glucose, likely through ROS, impairs the EGFR-phosphatidylinositol 3-kinase/Akt pathway, resulting in delayed corneal epithelial wound healing. Antioxidants in combination with EGFR ligands may be promising potential therapeutics for diabetic keratopathy.

Cross talk between c-Met and epidermal growth factor receptor during retinal pigment epithelial wound healing.

PURPOSE: The authors sought to determine how hepatocyte growth factor (HGF) receptor c-Met and epidermal growth factor receptor (EGFR) cross talk in response to injury in human ARPE-19 cells. METHODS: A scratch wound was made on a cell monolayer of ARPE-19 cells using a sequence-comb or a pipet tip, and it was allowed to heal in the presence or absence of HGF and heparin-binding EGF-like growth factor (HB-EGF). The activation of EGFR was analyzed by immunoprecipitation with EGFR antibody, followed by Western blotting with phosphotyrosine-specific antibody. Phosphorylation of extracellular signal-regulated kinase (ERK) and AKT (a major substrate of phosphatidylinositol 3'-kinase (PI3K) was assessed by Western blotting. The release of c-Met ectodomain into the culture media was determined by Western blotting using an antibody against the extracellular region. Cell migration was assessed by Boyden chamber migration assay. RESULTS: ARPE-19 cells underwent spontaneous wound healing in basal medium, and exogenously added HB-EGF and HGF significantly enhanced wound closure. Basal and growth factor-enhanced wound closures were attenuated but not slowed by hydroxyurea, a cell proliferation inhibitor. RPE cells expressed all four erbBs, and wounding induced EGFR transactivation and downstream ERK and PI3K phosphorylation in ARPE-19 cells. HGF also induced EGFR tyrosine phosphorylation. The EGFR kinase inhibitor AG1478 blocked wound- and HGF-stimulated EGFR transactivation and attenuated spontaneous and growth factor-induced wound closure. Wounding and EGFR ligands induced the release of c-Met into the culture media. Moreover, pretreatment of cells with HB-EGF impaired ARPE-19 migration toward HGF in a matrix metalloproteinase inhibitor-sensitive manner. CONCLUSIONS: EGFR modulates HGF/c-Met activity by inducing c-Met ectodomain shedding, and HGF/c-Met transactivates EGFR, leading to an enhanced activation of downstream signaling pathways. Cross talk between EGFR and c-Met may play a key role in regulating RPE cell migration, proliferation, and wound healing.

Wound-induced ATP release and EGF receptor activation in epithelial cells.

We have shown previously that wounding of human corneal epithelial (HCE) cells resulted in epidermal growth factor receptor (EGFR) transactivation through ectodomain shedding of heparin-binding EGF-like growth factor (HB-EGF). However, the initial signal to trigger these signaling events in response to cell injury remains elusive. In the present study, we investigated the role of ATP released from the injured cells in EGFR transactivation in HCE cells as well as in BEAS 2B cells, a bronchial epithelial cell line. Wounding of epithelial monolayer resulted in the release of ATP into the culture medium. The wound-induced rapid activation of phosphatidylinositol-3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) pathways in HCE cells was attenuated by eliminating extracellular ATP, ADP and adenosine. The nonhydrolyzable ATP analog ATP-gamma-S induced rapid and sustained EGFR activation that depended on HB-EGF shedding and ADAM (a disintegrin and metalloproteinase). Targeting pathways leading to HB-EGF shedding and EGFR activation attenuated ATP-gamma-S-enhanced closure of small scratch wounds. The purinoceptor antagonist reactive blue 2 decreased wound closure and attenuated ATP-gamma-S induced HB-EGF shedding. Taken together, our data suggest that ATP, released upon epithelial injury, acts as an early signal to trigger cell responses including an increase in HB-EGF shedding, subsequent EGFR transactivation and its downstream signaling, resulting in wound healing.