Passivating Injured Endothelium with Kinexins in Thrombolytic Therapy.

Without conjunctive administration of an anticoagulant, endothelial injury-induced thrombosis is resistant to thrombolysis and prone to re-thrombosis. We hypothesized that co-delivery of recombinant tissue plasminogen activator (rtPA) with annexin V-containing anticoagulants that specifically target the injured endothelium may passivate the thrombogenic elements of the vascular injury site and enhance rtPA-induced thrombolysis. In this study, the effects of conjunctive administration of Kinexins (Kunitz inhibitor-annexin V fusion proteins) with rtPA on thrombolysis were determined in vitro and in vivo. Thromboelastometry showed that both TAP-A (tick anticoagulant peptide-annexin V fusion protein; an inhibitor of factor Xa [FXa] and prothrombinase) and A-6L15 (annexin V-6L15 fusion protein; an inhibitor of tissue factor/FVIIa) exerted concentration-dependent (10-100 nM) effects on clot formation, with TAP-A being several folds more potent than A-6L15 in whole blood. Combination of TAP-A or A-6L15 with rtPA (1 µg/mL) led to decrease in lysis index, suggesting conjunctive enhancement of thrombolysis by combined use of rtPA with TAP-A or A-6L15. In a rat cremaster muscle preparation subjected to photochemical injury, conjunctive administration of rtPA and TAP-A significantly restored tissue perfusion to 56%, which is approximately two fold of that by rtPA or TAP-A alone. Near-infrared fluorescence images demonstrated local retention of a fluorescent A-6L15-S288 at the injury site, suggesting a targeting effect of the fusion protein. Pharmacokinetic analysis showed that 123I-labelled TAP-A and A-6L15 had initial distribution half-lives (T1/2α) of approximately 6 minutes and elimination half-lives (T1/2ß) of approximately 2.3 hours. In conclusion, Kinexins were potentially useful adjunctive agents with rtPA thrombolytic therapy especially for thrombosis induced by endothelial injury.

Anti-angiogenic effect of soybean kunitz trypsin inhibitor on human umbilical vein endothelial cells.

Soybean kunitz trypsin inhibitor (STI) was purified from aqueous extract of defatted soybean meal by affinity and ion exchange chromatography. In this study the effect of purified STI on cell migration and tubulogenesis in microcarrier-based fibrin gel was assayed. Purified STI had strong inhibitory effect on human umbilical vein endothelial cells migration and tubulogenesis in fibrin matrix, without toxic effects in the studied doses.

Overexpression of a hybrid gene consisting of the amino-terminal fragment of urokinase and carboxyl-terminal domain of bikunin suppresses invasion and migration of human ovarian cancer cells in vitro.

A Kunitz-type protease inhibitor, bikunin, is known to suppress the invasion and metastasis of cancer cells. HI8, a carboxyl-terminal domain of bikunin, is an active site of this glycoprotein. To increase its affinity for cancer cells, we constructed a chimeric gene, ATF-HI8, and investigated the anti-invasive and anti-migratory activity of ATF-HI8 on ovarian cancer cells. ATF-HI8-expressing plasmid and ATF-expressing plasmid were introduced into the highly invasive and metastatic ovarian cancer cell line HRA. The properties of the established cell line (HRA/ATF-HI8) were compared to those of the HRA/ATF and the HRA/luciferase (HRA/LUC, control) cell lines in terms of cell proliferation, invasion and migration. As a result, (i) there were no differences in cell proliferation between HRA/ATF-HI8 and HRA/LUC; (ii) the invasion and migration of HRA/ATF-HI8 cells were significantly inhibited compared to those of HRA/LUC cells; (iii) the migration, but not the invasion, of HRA/ATF cells was significantly inhibited compared to that of HRA/LUC. These results indicate that the overexpression of ATF-HI8 inhibits the invasion and migration of ovarian cancer cells without affecting cell proliferation and suggest that HI8 is involved in the anti-invasive and the anti-migratory activities, and the addition of ATF brought about the increase in the anti-migratory activity of HI8. The above findings suggest the applicability of therapeutic strategies targeting the inhibition of peritoneal invasion and dissemination of ovarian cancer by the use of the chimeric gene ATF-HI8.

Bikunin plus paclitaxel markedly reduces tumor burden and ascites in mouse model of ovarian cancer.

Our previous studies of intraperitoneal ovarian carcinoma in a mouse model demonstrated that bikunin gene transfection could prevent ascites formation and intraperitoneal disseminated metastasis. Although ascites was almost completely inhibited, tumor burden was variably reduced. Several reports have indicated that bikunin may be involved in tumor survival. In the present study, the effectiveness of exogenous bikunin and the biodistribution characteristics of (125)I-bikunin were initially examined in a mouse model of human ovarian cancer HRA cells. The once-daily i.p. administration of bikunin significantly decreased progressive growth of HRA tumors and ascites formation in a dose-dependent manner. Maximal radioisotope tumor uptake peaked at 7.4% injected dose/g at 3 hr. Bikunin binding specificity was demonstrated by reduced tumor uptake after coinjection of excess nonradioactive bikunin. Bikunin was rapidly excreted renally. The bikunin therapy produced the significant inhibition in expression of the proteolysis (uPA and uPAR) and angiogenesis-related molecules (VEGF and bFGF). The second purpose of our study was to optimize the antimetastatic activity of bikunin in combination with paclitaxel against HRA cells growing orthotopically in mice. The once-daily i.p. administration of bikunin (25 microg/g body weight/day) in combination with paclitaxel (i.p., 100 microg/20 g at days 2 and 5) significantly decreased progressive growth of HRA cells in a synergistic fashion. In conclusion, combination therapy with bikunin plus paclitaxel may be an effective way to markedly reduce i.p. tumor growth and ascites in ovarian carcinoma possibly through suppression of uPA, uPAR, VEGF and bFGF expression.

Soybean trypsin inhibitor confers protection against rotavirus infection in infant mice.

OBJECTIVE: To study the effects of soybean trypsin inhibitor (TI) on glycine uptake, glutathione (GSH) levels and morphological changes of intestine in rotavirus (RV) infected infant mice. METHODS: A total of 144 infant mice (7/8 days old) were divided in 3 groups (i.e. control, RV and RV + inhibitor). Infant mice were orally inoculated with the EB strain of RV and Trypsin protease inhibitor (TI) and 8 animals each were sacrificed on days 0,1,3,5,7 and 10 post infection (p.i). Glycine uptake (in vitro), GSH levels and histological changes were assessed in the jejunum, ileum and colon. RESULTS: Glycine uptake and GSH levels were significantly reduced on days 3 and 5 p.i in jejunum and ileum of RV inoculated animals, compared to the controls. Glycine uptake and GSH levels were maintained as in controls in the RV + TI inoculated animals on days 3 and 5 p.i in jejunum and colon but not in ileum where lesser values were recorded. Histology showed vacuolar degeneration in ileum towards the apical portion whereas normal morphology was observed in jejunum, similar to controls. No histological changes were observed in colon in any of the groups. Electron microscopic study confirmed the viral infection. CONCLUSION: Administration of Trypsin protease inhibitor along with RV reverted the effects of RV infection on amino acid uptake and GSH levels completely in the jejunum and partially in the ileum.

Influence of soybean trypsin inhibitor on small bowel enzyme activities during rotavirus infection in malnourished infant mice.

Rotavirus (RV) is considered to be one of the major causes of acute episodic diarrhoea throughout the world. This study was undertaken to investigate the effect of soybean trypsin inhibitor (TI) on brush-border enzymes during rotavirus infection in protein energy malnourished (PEM) infant mice. Animals were divided into 4 groups, namely controls, PEM, PEM+RV and PEM+RV+TI (n = 36 each). Group 1 and 2 animals were orally inoculated with 50 microl of normal saline each. Group 3 animals were orally inoculated with 50 microl of 100 ID50 dose of RV stock each. Group 4 animals were similarly inoculated with 0.6 mg TI/g body weight along with 50 microl of RV stock each. Animals were examined daily for diarrhoea and their body weight was recorded on alternate days postinoculation (dpi). Animals were killed by cervical dislocation after being given light chloroform anesthesia on 0, 1, 3, 5, 7 and 10 dpi. Small intestines were excised and homogenized in normal saline. Proteins, gammaglutamyl transpeptidase, alkaline phosphatase and disaccharidases were estimated in jejunum and ileum. Body weight was significantly reduced in PEM animals and with RV infection. Histologically, focal areas of vacuolar degeneration of lining epithelium were seen in RV-infected animals. Disaccharidases and other enzyme activities were decreased significantly in the PEM group compared to healthy controls and further depressed with RV infection in malnourished animals as compared to non-infected PEM. The enzyme activities were restored in animals receiving TI along with RV compared to the group receiving RV without TI. With the administration of soybean TI, the activities of disaccharidases, alkaline phosphatase, gammaglutamyl transpeptidase and intestinal architecture were restored showing a protective effect in PEM during RV infection.

Protective efficacy of trypsin inhibitor on the the gut following rotavirus infection in malnourished infant mice.

Epidemiologic data suggest a close relationship between protein energy malnutrition (or protein energy malnourished) (PEM) and development of infectious diarrheal diseases. In the present studies, the impact of trypsin inhibitor (TI) on the gut following rotavirus (RV) infection was investigated in PEM young growing inbred balb/c mice. The animals were divided into four groups: (1) control; (2) PEM; (3) PEM+RV and (4) PEM+ RV+TI. Control and PEM animals were orally inoculated with normal saline, PEM+RV animals were given 50 microl RV (100ID50) and PEM+RV+TI animals were inoculated with 0.6 mg TI/g body weight + 50 micronl RV. The body weight of each animal was recorded. Six animals from each group were sacrificed on days 0, 1, 3, 5, 7 and 10 post inoculation by cervical dislocation. The intestines were removed, everted and homogenized in ice-cold saline. Leucine aminopeptidase (LAP), glutathione (GSH) levels and L-leucine uptake were determined in the jejunum and ileum. Body weight was decreased in PEM and PEM+RV groups compared to the controls. LAP and leucine uptake levels were elevated in the PEM group but decreased in the PEM+RV group. GSH levels were decreased in the PEM+RV group. Enzyme activity, GSH and LAP uptake levels were restored in the PEM+RV+TI group. The study shows the protective role of soybean TI against RV infection in malnourished mice.

Neutrophil-dependent decrease in early wound margin strength.

We evaluated the effect of neutropenia or administration of a serine proteinase inhibitor on the early suture-holding capacity of intestinal anastomoses in rats. One group of rats was treated with antineutrophil serum, and another group received the soybean trypsin inhibitor. Controls received inactivated serum or saline. Anastomotic suture-holding capacity (breaking strength), myeloperoxidase activity, and collagen were measured 0 and 72 hours after surgery. Suture-holding capacity decreased by 70% in controls and 35% in soybean trypsin inhibitor-treated rats, but remained on level with immediate postoperative strength in neutropenic rats, where low myeloperoxidase levels reflected effective wound margin neutropenia. Collagen content and solubility were similar in all groups. These findings indicate that reduction in early wound margin strength is neutrophil dependent, and that neutrophil serine proteinases are important mediators in that process.

Sucralfate in the prevention of acute gastric lesions induced by ischemia-reperfusion

En ratas Wistar, se estudió el rol del Sucralfato en la prevención de las lesiones agudas gástricas, en el modelo de la isquemia-reperfusion por oclusión total del tronco celiaco y su comparación con bloqueadores de los radicales libres, como el Alopurinol, el Inhibidor de la Tripsina de Soja y la Superóxido Dismutasa. En ratas controles, el área necrótica macroscópica de la mucosa gástrica fue de un 80%; en cambio, las drogas antioxidantes dieron un área necróticia entre el 7 al 15% y Sucralfato dio escasamente un 4%. Se concluyó que Sucralfato como ciroprotector y antioxidante, al incrementar la barrera defensiva gástrica fue más importante que la agresión secundaria de los radicales libres

Sucralfate in the prevention of acute gastric lesions induced by ischemia-reperfusion

En ratas Wistar, se estudió el rol del Sucralfato en la prevención de las lesiones agudas gástricas, en el modelo de la isquemia-reperfusion por oclusión total del tronco celiaco y su comparación con bloqueadores de los radicales libres, como el Alopurinol, el Inhibidor de la Tripsina de Soja y la Superóxido Dismutasa. En ratas controles, el área necrótica macroscópica de la mucosa gástrica fue de un 80%; en cambio, las drogas antioxidantes dieron un área necróticia entre el 7 al 15% y Sucralfato dio escasamente un 4%. Se concluyó que Sucralfato como ciroprotector y antioxidante, al incrementar la barrera defensiva gástrica fue más importante que la agresión secundaria de los radicales libres (AU)

Sucralfate in the prevention of acute gastric lesions induced by ischemia-reperfusion.

The role of Sucralfate in prevention of acute gastric injuries and its comparison with free radicals blockers as Allopurinol, Soybean Trypsin Inhibitor and Superoxide Dismutase was studied in the ischemia-reperfusion model by total occlusion of the celiac axis in Wistar rats. In control rats, the gross gastric mucosal necrotic area was of 80%; in contrast, the antioxidant drugs resulted in a necrotic area of 7%-15% and Sucralfate resulted in a necrotic area of only a 4%. It was concluded that Sucralfate, as antioxidant-cytoprotective drug, by enhancing the gastric defensive barrier was more important than the secondary aggression induced by free radicals.

Soybean trypsin inhibitor attenuates ischemic injury to the feline small intestine.

Recent evidence suggests that oxygen free radicals are largely responsible for the increased vascular permeability and early mucosal lesions associated with partial intestinal ischemia. It is postulated that oxygen radicals are produced by the reaction of the enzyme xanthine oxidase with hypoxanthine and molecular oxygen. In normal healthy cells, xanthine oxidase exists as a nicotinamide adenine dinucleotide-reducing dehydrogenase and not the oxygen radical-producing oxidase. In the intestine, dehydrogenase-to-oxidase conversion is nearly complete with less than 1 min of ischemia. Biochemical evidence from the intestine and liver indicate that ischemia-induced conversion of xanthine dehydrogenase to xanthine oxidase can be prevented by administration of protease inhibitors such as soybean trypsin inhibitor. In order to assess the role of proteases in oxygen radical-mediated ischemic injury to the small bowel, quantitative analyses of mucosal lesion development and vascular permeability were performed in autoperfused segments of cat ileum subjected to 1 or 3 h of ischemia and pretreated with 15 mg/kg (i.v.) soybean trypsin inhibitor. One hour of ischemia produced a significant increase in intestinal vascular permeability. The ischemia-induced increase in vascular permeability was significantly attenuated by soybean trypsin inhibitor pretreatment. Three hours of ischemia led to the development of mucosal lesions in untreated animals. Pretreatment with soybean trypsin inhibitor largely prevented the development of the mucosal lesions. The findings of our study are consistent with biochemical evidence that, during ischemia, proteases trigger the conversion of xanthine dehydrogenase to xanthine oxidase and thereby lead to oxygen radical production and subsequent tissue injury.