The purpose of this study was to investigate the correlations between endothelial cell loss rate at one week, one month, three months, and six months after penetrating keratoplasty and clinical factors including recipient age, donor age, preoperative endothelial cell densities of the donors and preoperative diagnoses of the recipients. Among the 70 patients who underwent penetrating keratoplasty at Chonbuk University Hospital from December 1996 to January 1999, 30 eyes of 30 patients that showed reasonable endothelial resolution by non-contact specular microscopy during the follow up period of 6 months were chosen and evaluated.The cell density decreased continuously during the observation period. Endothelial cell loss rate averaged 11.5%at one week, 19.0% at one month, 25.0%at three months and 30.0%at six months after penetrating keratoplasty. Endothelial cell loss rate showed no significant correlation with donor age, recipient age, preoperative cell density at any examination period[r+/-0.4, p0.05]. Preoperative endothelial cell density showed significant correlation with postoperative endothelial cell density at each examination period[r0.7, p<0.05]. The rate of endothelial loss in the keratoconus group was significantly lower than those of bullous keratopathy or corneal leukoma groups at three months after penetrating keratoplasty[Wilcoxon test, p<0.05].
Cell Count ; Corneal Opacity ; Diagnosis ; Endothelial Cells* ; Follow-Up Studies ; Humans ; Jeollabuk-do ; Keratoconus ; Keratoplasty, Penetrating* ; Microscopy ; Tissue Donors

BACKGROUND: Graft vessel preservation solution in coronary artery bypass surgery is used to maintain the graft conduit in optimal condition during the perioperative period. Nafamostat mesilate (NM) has anticoagulation and anti-inflammatory properties. Therefore, we investigated NM as a conduit preservative agent and compared it to papaverine. METHODS: Sprague-Dawley (SD) rat thoracic aortas were examined for their contraction-relaxation ability using phenylephrine (PE) and acetylcholine (ACh) following preincubation with papaverine and NM in standard classical organ baths. Human umbilical vein endothelial cells (HUVECs) were cultured to check for the endothelial cell viability. Histopathological examination and terminal deoxynucleotidyl transferase dUTP nick end labeling assay were performed on the thoracic aortas of SD rats. RESULTS: The anti-contraction effects of papaverine were superior to those of NM at PE (p<0.05). The relaxation effect of NM on ACh-induced vasodilatation was not statistically different from that of papaverine. Viability assays using HUVECs showed endothelial cell survival rates of >90% in various concentrations of both NM and papaverine. A histopathological study showed a protective effect against necrosis and apoptosis (p<0.05) in the NM group. CONCLUSION: NM exhibited good vascular relaxation and a reasonable anti-vasocontraction effect with a better cell protecting effect than papaverine; therefore, we concluded that NM is a good potential conduit preserving agent.
Acetylcholine ; Animals ; Anticoagulants ; Aorta, Thoracic ; Apoptosis ; Baths ; Coronary Artery Bypass* ; Coronary Vessels* ; DNA Nucleotidylexotransferase ; Endothelial Cells ; Guanidines ; Human Umbilical Vein Endothelial Cells ; Mesylates* ; Necrosis ; Papaverine ; Perioperative Period ; Phenylephrine ; Rats ; Rats, Sprague-Dawley ; Relaxation ; Survival Rate ; Transplants ; Vasodilation

BACKGROUND: Unfractionated heparin is commonly used for anticoagulation in extracorporeal membrane oxygenation (ECMO). Several studies have shown that nafamostat mesilate (NM) has comparable clinical outcomes to unfractionated heparin. This study compared anticoagulation with NM and heparin in a large-animal model. METHODS: Beagle dogs (n=8; weight, 6.5–9 kg) were placed on venovenous ECMO. Blood samples were taken every hour and the following parameters were compared: hemoglobin level, activated partial thromboplastin time (aPTT), thromboelastography (TEG) data, platelet function, and inflammatory cytokine levels. RESULTS: In both groups, the aPTT was longer than the baseline value. Although the aPTT in the NM group was shorter than in the heparin group, the TEG parameters were similar between the 2 groups. Hemoglobin levels decreased in both groups, but the decrease was less with NM than with heparin (p=0.049). Interleukin (IL)-1β levels significantly decreased in the NM group (p=0.01), but there was no difference in the levels of tumor necrosis factor alpha or IL-10 between the 2 groups. CONCLUSION: NM showed a similar anticoagulant effect to that of unfractionated heparin, with fewer bleeding complications. NM also had anti-inflammatory properties during ECMO. Based on this preclinical study, NM may be a good alternative candidate for anticoagulation in ECMO.
Animals ; Anticoagulants ; Blood Platelets ; Dogs ; Extracorporeal Membrane Oxygenation* ; Hemorrhage ; Heparin ; Interleukin-10 ; Interleukins ; Mesylates* ; Partial Thromboplastin Time ; Thrombelastography ; Tumor Necrosis Factor-alpha

Arterial thrombosis and its associated diseases are considered to constitute a major healthcare problem. Arterial thrombosis, defined as blood clot formation in an artery that interrupts blood circulation, is associated with many cardiovascular diseases. Oxidative stress is one of many important factors that aggravates the pathophysiological process of arterial thrombosis. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (Ref-1) has a multifunctional role in cells that includes the regulation of oxidative stress and anti-inflammatory function. The aim of this study was to investigate the therapeutic effect of adenovirus-mediated Ref-1 overexpression on arterial thrombosis induced by 60% FeCl3 solution in rats. Blood flow was measured to detect the time to occlusion, thrombus formation was detected by hematoxylin and eosin staining, reactive oxygen species (ROS) levels were detected by high-performance liquid chromatography, and the expression of tissue factor and other proteins was detected by Western blot. FeCl3 aggravated thrombus formation in carotid arteries and reduced the time to artery occlusion. Ref-1 significantly delayed arterial obstruction via the inhibition of thrombus formation, especially by downregulating tissue factor expression through the Akt-GSK3β-NF-κB signaling pathway. Ref-1 also reduced the expression of vascular inflammation markers ICAM-1 and VCAM-1, and reduced the level of ROS that contributed to thrombus formation. The results showed that adenovirus-mediated Ref-1 overexpression reduced thrombus formation in the rat carotid artery. In summary, Ref-1 overexpression had anti-thrombotic effects in a carotid artery thrombosis model and could be a target for the treatment of arterial thrombosis.

Arterial thrombosis and its associated diseases are considered to constitute a major healthcare problem. Arterial thrombosis, defined as blood clot formation in an artery that interrupts blood circulation, is associated with many cardiovascular diseases. Oxidative stress is one of many important factors that aggravates the pathophysiological process of arterial thrombosis. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (Ref-1) has a multifunctional role in cells that includes the regulation of oxidative stress and anti-inflammatory function. The aim of this study was to investigate the therapeutic effect of adenovirus-mediated Ref-1 overexpression on arterial thrombosis induced by 60% FeCl3 solution in rats. Blood flow was measured to detect the time to occlusion, thrombus formation was detected by hematoxylin and eosin staining, reactive oxygen species (ROS) levels were detected by high-performance liquid chromatography, and the expression of tissue factor and other proteins was detected by Western blot. FeCl3 aggravated thrombus formation in carotid arteries and reduced the time to artery occlusion. Ref-1 significantly delayed arterial obstruction via the inhibition of thrombus formation, especially by downregulating tissue factor expression through the Akt-GSK3β-NF-κB signaling pathway. Ref-1 also reduced the expression of vascular inflammation markers ICAM-1 and VCAM-1, and reduced the level of ROS that contributed to thrombus formation. The results showed that adenovirus-mediated Ref-1 overexpression reduced thrombus formation in the rat carotid artery. In summary, Ref-1 overexpression had anti-thrombotic effects in a carotid artery thrombosis model and could be a target for the treatment of arterial thrombosis.

To evaluate the involvement of nitric oxide production on the endothelium-dependent relaxation in diabetes, we have measured vascular and endothelial function and nitric oxide concentration, and the expression level of endothelial nitric oxide synthase in the streptozotocin-induced diabetic rats. Diabetic rats were induced by the injection of streptozotocin (50 mg/kg i.v.) in the Sprague-Dawley rats. Vasoconstrictor responses to nonrepinephrine (NE) showed that maximal contraction to norepinephrine (10(-5) M) was significantly enhanced in the aorta of diabetic rats. Endothelium-dependent relaxation induced by acetylcholine was markedly impaired in the aorta of diabetic rats, these responses were little improved by the pretreatment with indomethacin. However, endothelium-independent relaxation induced by nitroprusside was not altered in the diabetic rats. Plasma nitrite and nitrate (NO2/3) levels in diabetic rats were significantly lower than innon-diabetic rats. Western blot analysis using a monoclonal antibody against endothelial cell nitric oxide synthase (eNOS) revealed that the protein level was lower in the aorta of diabetic rats than in non-diabetic rats. These data indicate that nitric oxide formation and eNOS expression is reduced in diabetes, and this would, in part, account for the impaired endothelium-dependent relaxation in the aorta of streptozotocin-induced diabetic rats.
Acetylcholine ; Animals ; Aorta ; Blotting, Western ; Endothelial Cells ; Indomethacin ; Nitric Oxide Synthase ; Nitric Oxide Synthase Type III ; Nitric Oxide* ; Nitroprusside ; Norepinephrine ; Plasma ; Rats* ; Rats, Sprague-Dawley ; Relaxation* ; Streptozocin

Among the Shc proteins, p66shc is known to be related to oxidative stress responses and regulation of the production of reactive oxygen species (ROS). The present study was undertaken to investigate the role of p66shc on endothelial nitric oxide synthase (eNOS) activity in the mouse embryonic fibroblasts (MEFs). When wild type (WT) or p66shc (-/-) MEFs were transfected with full length of eNOS cDNA, the expression and activity of eNOS protein were higher in the p66shc (-/-) MEFs. These phenomena were reversed by reconstitution of p66shc cDNA transfection in the p66shc (-/-) MEFs. The basal superoxide production in the p66shc (-/-) MEFs was not significantly different from that of WT of MEFs. However, superoxide production induced by NADPH in the p66shc (-/-) MEF was lesser than that in WT MEFs. When compared with WT MEFs, cell lysate of p66shc (-/-) MEFs showed significantly increased H-ras activity without change of endogenous H-ras expression. Our findings suggest the pivotal role of p66shc adaptor protein played in inhibition of endothelial nitric oxide production via modulation of the expression and/or activity of eNOS protein.
Animals ; DNA, Complementary ; Endothelium ; Fibroblasts* ; Mice* ; NADP ; Nitric Oxide ; Nitric Oxide Synthase Type III* ; Oxidative Stress ; Reactive Oxygen Species ; Superoxides ; Transfection

Purpose: The isocitrate dehydrogenase (IDH) family plays an essential role in metabolism and energy production. The relative expression levels of IDH isoforms (IDH1, IDH2, and IDH3) have prognostic significance in several malignancies, including breast carcinoma. However, the IDH isozyme expression levels in different cancer stages and types have not been determined in breast carcinoma tissues. Methods: We analyzed the messenger RNA (mRNA) and protein levels of IDH (IDH1, IDH2, and IDH3A) and α-ketoglutarate (α-KG) in 59 breast carcinoma tissues. Results: The mRNA level of IDH2 was significantly increased at stages 2 and 3 in triple-negative and (ER-/PR-/HER+) breast cancers. However, the elevated α-KG level was only observed in stages 2 and 3, with no differences in the various breast carcinoma types. Western blotting analysis showed that IDH2 protein expression increased in the patient tissues and cell lines. An in vitro study showed IDH2 downregulation in the triple-negative breast cancer cell line MDA-MB-231 that inhibited cell proliferation and migration and induced cell cycle arrest in the G0/G1 phase. Conclusion These findings suggest that different from IDH1 and IDH3, IDH2 is more highly expressed in stages 2 and 3 breast cancer tissues, especially in triple-negative breast cancer. IDH2 potentially serves as a target to detect unknown mechanisms in breast cancer.

Schwann cells are the most abundant cells in the peripheral nervous system, maintaining the development, function and regeneration of peripheral nerves. Defects in these Schwann cells injury response potentially contribute to the pathogenesis of diabetic peripheral neuropathy (DPN), a common complication of diabetes mellitus. The protein p66shc is essential in regulating oxidative stress responses, autophagy induction and cell survival, and is also vital in the development of DPN. In this study, we hypothesized that p66shc mediates high glucose-induced oxidative stress and autophagic dysfunction. In Schwann cells treated with high glucose; p66shc expression, levels of reactive oxygen species, autophagy impairment, and early apoptosis were elevated. Inhibition of p66shc gene expression by siRNA reversed high glucose-induced oxidative stress, autophagy impairment, and early apoptosis. We also demonstrated that the levels of p66shc was increased, while autophagy-related proteins p62 and LC3 (LC3-II/I) were suppressed in the sciatic nerve of streptozotocin-induced diabetes mice. P66shc-deficient mice exhibited the improvement in autophagy impairment after diabetes onset. Our findings suggest that the p66 plays a crucial role in Schwann cell dysfunction, identifying its potential as a therapeutic target.

The role of acetylated apurinic/apyrimidinic endonuclease 1/redox factor 1 (APE1/Ref-1) in ovarian cancer remains poorly understood. Therefore, this study aimed to investigate the combined effect of recombinant human APE1/Ref-1 (rhAPE1/Ref-1) and aspirin (ASA) on two ovarian cancer cells, PEO-14, and CAOV3.The viability and apoptosis of ovarian cancer cells treated with rhAPE1/Ref-1 or ASA were assessed. Our results demonstrated that ASA induced rhAPE1/Ref-1 acetylation and widespread hyperacetylation in PEO-14 cells. Additionally, co-treatment with rhAPE1/Ref-1 and ASA substantially reduced cell viability and induced PEO-14 cell apoptosis, not CAOV3, in a dose-dependent manner. ASA increased the expression and membrane localization of the receptor for advanced glycation endproducts (RAGEs). Acetylated APE1/Ref-1 showed enhanced binding to RAGEs. In contrast, RAGE knockdown reduced cell death and poly(ADP-ribose) polymerase cleavage caused by rhAPE1/Ref-1 and ASA combination treatment, highlighting the importance of the APE1/Ref-1-RAGE interaction in triggering apoptosis. Moreover, combination treatment with rhAPE1/Ref-1 and ASA effectively induced apoptosis in 3D spheroid cultures of PEO-14 cells, a model that better mimics the tumor microenvironment. These results demonstrate that acetylated APE1/Ref-1 and its interaction with RAGE is a potential therapeutic target for ovarian cancer. Thus, the combination of ASA and APE1/Ref-1 may offer a promising new strategy for inducing cancer cell death.