Ref-1 protects against FeCl3 -induced thrombosis and tissue factor expression via the GSK3β–NF-κB pathway

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.

Ref-1 protects against FeCl3 -induced thrombosis and tissue factor expression via the GSK3β–NF-κB pathway

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.

Validation of Nafamostat Mesilate as an Anticoagulant in Extracorporeal Membrane Oxygenation: A Large-Animal Experiment

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.

Protein kinase C beta II upregulates intercellular adhesion molecule-1 via mitochondrial activation in cultured endothelial cells

Activation of protein kinase C (PKC) is closely linked with endothelial dysfunction. However, the effect of PKCβII on endothelial dysfunction has not been characterized in cultured endothelial cells. Here, using adenoviral PKCβII gene transfer and pharmacological inhibitors, the role of PKCβII on endothelial dysfucntion was investigated in cultured endothelial cells. Phorbol 12-myristate 13-acetate (PMA) increased reactive oxygen species (ROS), p66shc phosphorylation, intracellular adhesion molecule-1, and monocyte adhesion, which were inhibited by PKCβi (10 nM), a selective inhibitor of PKCβII. PMA increased the phosphorylation of CREB and manganese superoxide dismutase (MnSOD), which were also inhibited by PKCβi. Gene silencing of CREB inhibited PMA-induced MnSOD expression, suggesting that CREB plays a key role in MnSOD expression. Gene silencing of PKCβII inhibited PMA-induced mitochondrial ROS, MnSOD, and ICAM-1 expression. In contrast, overexpression of PKCβII using adenoviral PKCβII increased mitochondrial ROS, MnSOD, ICAM-1, and p66shc phosphorylation in cultured endothelial cells. Finally, PKCβII-induced ICAM-1 expression was inhibited by Mito-TEMPO, a mitochondrial ROS scavenger, suggesting the involvement of mitochondrial ROS in PKC-induced vascular inflammation. Taken together, the results suggest that PKCβII plays an important role in PMA-induced endothelial dysfunction, and that the inhibition of PKCβII-dependent p66shc signaling acts as a therapeutic target for vascular inflammatory diseases.

Nafamostat mesilate promotes endothelium-dependent vasorelaxation via the Akt-eNOS dependent pathway

Nafamostat mesilate (NM), a synthetic serine protease inhibitor, has anticoagulant and anti-inflammatory properties. The intracellular mediator and external anti-inflammatory external signal in the vascular wall have been reported to protect endothelial cells, in part due to nitric oxide (NO) production. This study was designed to examine whether NM exhibit endothelium dependent vascular relaxation through Akt/endothelial nitric oxide synthase (eNOS) activation and generation of NO. NM enhanced Akt/eNOS phosphorylation and NO production in a dose- and time-dependent manner in human umbilical vein endothelial cells (HUVECs) and aorta tissues obtained from rats treated with various concentrations of NM. NM concomitantly decreased arginase activity, which could increase the available arginine substrate for NO production. Moreover, we investigated whether NM increased NO bioavailability and decreased aortic relaxation response to an eNOS inhibitor in the aorta. These results suggest that NM increases NO generation via the Akt/eNOS signaling pathway, leading to endothelium-dependent vascular relaxation. Therefore, the vasorelaxing action of NM may contribute to the regulation of cardiovascular function.

In Vivo Neuroprotective Effect of Histidine-Tryptophan-Ketoglutarate Solution in an Ischemia/Reperfusion Spinal Cord Injury Animal Model

BACKGROUND: Paraplegia is a devastating complication following operations on the thoracoabdominal aorta. We investigated whether histidine-tryptophan-ketoglutarate (HTK) solution could reduce the extent of ischemia/reperfusion (IR) spinal cord injuries in a rat model using a direct delivery method. METHODS: Twenty-four Sprague-Dawley male rats were randomly divided into four groups. The sham group (n=6) underwent a sham operation, the IR group (n=6) underwent only an aortic occlusion, the saline infusion group (saline group, n=6) underwent an aortic occlusion and direct infusion of cold saline into the occluded aortic segment, and the HTK infusion group (HTK group, n=6) underwent an aortic occlusion and direct infusion of cold HTK solution into the occluded aortic segment. An IR spinal cord injury was induced by transabdominal clamping of the aorta distally to the left renal artery and proximally to the aortic bifurcation for 60 minutes. A neurological evaluation of locomotor function was performed using the modified Tarlov score after 48 hours of reperfusion. The spinal cord was harvested for histopathological and immunohistochemical examinations. RESULTS: The spinal cord IR model using direct drug delivery in rats was highly reproducible. The Tarlov score was 4.0 in the sham group, 1.17±0.75 in the IR group, 1.33±1.03 in the saline group, and 2.67±0.81 in the HTK group (p=0.04). The histopathological analysis of the HTK group showed reduced neuronal cell death. CONCLUSION: Direct infusion of cold HTK solution into the occluded aortic segment may reduce the extent of spinal cord injuries in an IR model in rats.

Nafamostat Mesilate Inhibits TNF-alpha-Induced Vascular Endothelial Cell Dysfunction by Inhibiting Reactive Oxygen Species Production

Nafamostat mesilate (NM) is a serine protease inhibitor with anticoagulant and anti-inflammatory effects. NM has been used in Asia for anticoagulation during extracorporeal circulation in patients undergoing continuous renal replacement therapy and extra corporeal membrane oxygenation. Oxidative stress is an independent risk factor for atherosclerotic vascular disease and is associated with vascular endothelial function. We investigated whether NM could inhibit endothelial dysfunction induced by tumor necrosis factor-alpha (TNF-alpha). Human umbilical vein endothelial cells (HUVECs) were treated with TNF-alpha for 24 h. The effects of NM on monocyte adhesion, vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) protein expression, p38 mitogen-activated protein kinase (MAPK) activation, and intracellular superoxide production were then examined. NM (0.01~100 microg/mL) did not affect HUVEC viability; however, it inhibited the increases in reactive oxygen species (ROS) production and p66shc expression elicited by TNF-alpha (3 ng/mL), and it dose dependently prevented the TNF-alpha-induced upregulation of endothelial VCAM-1 and ICAM-1. In addition, it mitigated TNF-alpha-induced p38 MAPK phosphorylation and the adhesion of U937 monocytes. These data suggest that NM mitigates TNF-alpha-induced monocyte adhesion and the expression of endothelial cell adhesion molecules, and that the anti-adhesive effect of NM is mediated through the inhibition of p66shc, ROS production, and p38 MAPK activation.

Trichostatin A Modulates Angiotensin II-induced Vasoconstriction and Blood Pressure Via Inhibition of p66shc Activation

Histone deacetylase (HDAC) has been recognized as a potentially useful therapeutic target for cardiovascular disorders. However, the effect of the HDAC inhibitor, trichostatin A (TSA), on vasoreactivity and hypertension remains unknown. We performed aortic coarctation at the inter-renal level in rats in order to create a hypertensive rat model. Hypertension induced by abdominal aortic coarctation was significantly suppressed by chronic treatment with TSA (0.5 mg/kg/day for 7 days). Nicotinamide adenine dinucleotide phosphate-driven reactive oxygen species production was also reduced in the aortas of TSA-treated aortic coarctation rats. The vasoconstriction induced by angiotensin II (Ang II, 100 nM) was inhibited by TSA in both endothelium-intact and endothelium-denuded rat aortas, suggesting that TSA has mainly acted in vascular smooth muscle cells (VSMCs). In cultured rat aortic VSMCs, Ang II increased p66shc phosphorylation, which was inhibited by the Ang II receptor type I (AT1R) inhibitor, valsartan (10 microM), but not by the AT2R inhibitor, PD123319. TSA (1~10 microM) inhibited Ang II-induced p66shc phosphorylation in VSMCs and in HEK293T cells expressing AT1R. Taken together, these results suggest that TSA treatment inhibited vasoconstriction and hypertension via inhibition of Ang II-induced phosphorylation of p66shc through AT1R.

Nafamostat Mesilate: Can It Be Used as a Conduit Preserving Agent in Coronary Artery Bypass Surgery?

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 (p90% 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.

Tat-Mediated p66shc Transduction Decreased Phosphorylation of Endothelial Nitric Oxide Synthase in Endothelial Cells

We evaluated the role of Tat-mediated p66shc transduction on the activation of endothelial nitric oxide synthase in cultured mouse endothelial cells. To construct the Tat-p66shc fusion protein, human full length p66shc cDNA was fused with the Tat-protein transduction domain. Transduction of TAT-p66shc showed a concentration- and time-dependent manner in endothelial cells. Tat-mediated p66shc transduction showed increased hydrogen peroxide and superoxide production, compared with Tat-p66shc (S/A), serine 36 residue mutant of p66shc. Tat-mediated p66shc transduction decreased endothelial nitric oxide synthase phosphorylation in endothelial cells. Furthermore, Tat-mediated p66shc transduction augmented TNF-alpha-induced p38 MAPK phosphorylation in endothelial cells. These results suggest that Tat-mediated p66shc transduction efficiently inhibited endothelial nitric oxide synthase phosphorylation in endothelial cells.

Redox Factor-1 Inhibits Cyclooxygenase-2 Expression via Inhibiting of p38 MAPK in the A549 Cells

In this study, we evaluated the role of apurinic/apyrimidinic endonuclease1/redox factor-1 (Ref-1) on the tumor necrosis factor-alpha (TNF-alpha) induced cyclooxygenase-2 (COX-2) expression using A549 lung adenocarcinoma cells. TNF-alpha induced the expression of COX-2 in A549 cells, but did not induce BEAS-2B expression. The expression of COX-2 in A549 cells was TNF-alpha dose-dependent (5~100 ng/ml). TNF-alpha-stimulated A549 cells evidenced increased Ref-1 expression in a dose-dependent manner. The adenoviral transfection of cells with AdRef-1 inhibited TNF-alpha-induced COX-2 expression relative to that seen in the control cells (Ad beta gal). Pretreatment with 10 micrometer of SB203580 suppressed TNF-alpha-induced COX-2 expression, thereby suggesting that p38 MAPK might be involved in COX-2 expression in A549 cells. The phosphorylation of p38 MAPK was increased significantly after 5 minutes of treatment with TNF-alpha, reaching a maximum level at 10 min which persisted for up to 60 min. However, p38MAPK phosphorylation was markedly suppressed in the Ref-1-overexpressed A549 cells. Taken together, our results appear to indicate that Ref-1 negatively regulates COX-2 expression in response to cytokine stimulation via the inhibition of p38 MAPK phosphorylation. In the lung cancer cell lines, Ref-1 may be involved as an important negative regulator of inflammatory gene expression.

p66shc Adaptor Protein Suppresses the Activation of Endothelial Nitric Oxide Synthase in Mouse Embryonic Fibroblasts

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.

NADPH Oxidase and Mitochondrial ROS are Involved in the TNF-alpha-induced Vascular Cell Adhesion Molecule-1 and Monocyte Adhesion in Cultured Endothelial Cells

Atherosclerosis is considered as a chronic inflammatory process. However, the nature of the oxidant signaling that regulates monocyte adhesion and its underlying mechanism is poorly understood. We investigated the role of reactive oxygen species on the vascular cell adhesion molecule-1 (VCAM-1) and monocyte adhesion in the cultured endothelial cells. TNF-alpha at a range of 1~30 ng/ml induced VCAM-1 expression dose-dependently. BCECF-AM-labeled U937 cells firmly adhered on the surface of endothelial cells when the endothelial cells were incubated with TNF-alpha (15 ng/ml). Ten micromol/L of SB203580, an inhibitor of p38 MAPK, significantly reduced TNF-alpha-induced VCAM-1 expression, compared to the JNK inhibitor (40micromol/L of SP60015) or ERK inhibitor (40micrommol/L of U0126). Also, SB203580 significantly inhibited TNF-alpha-induced monocyte adhesion in HUVEC. Superoxide production was minimal in the basal condition, however, treatment of TNF-alpha induced superoxide production in the dihydroethidine-loaded endothelial cells. Diphenyleneiodonium (DPI, 10micromol/L), an inhibitor of NADPH oxidase, and rotenone (1micromol/L), an inhibitor of mitochondrial complex I inhibited TNF-alpha-induced superoxide production, VCAM-1 expression and monocyte adhesion in the endothelial cells. Taken together, our data suggest that NADPH oxidase and mitochondrial ROS were involved in TNF-alpha-induced VCAM-1 and monocyte adhesion in the endothelial cells.

Impaired endothelium-dependent relaxation is mediated by reduced production of nitric oxide in the streptozotocin-induced diabetic rats

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.