Naohiro Kanayama

Interleukin-8 induces cervical ripening in rabbits

OBJECTIVEnThe purpose of this study was to determine whether cervical ripening can be induced in rabbits by interleukin-8.nnnSTUDY DESIGNnNonpregnant and pregnant rabbits were treated for 5 days with vaginal suppositories containing 100 ng of interleukin-8. Collagen and glycosaminoglycan concentration in the cervices were assessed histologically by picrosirius red and alcian blue, and the mean optical density was calculated. The mean neutrophil count in five random fields was calculated from each biopsy specimen.nnnRESULTSnInterleukin-8 induced softening and dilatation of the rabbit cervices. Water content was significantly increased (p < 0.0001 and p < 0.001, respectively). Cervical collagen concentration was found to be significantly decreased (p < 0.0004 and p < 0.0001, respectively). Glycosaminoglycan concentration was significantly increased in nonpregnant and pregnant cervices (p < 0.0009 and p < 0.1, respectively). The mean number of neutrophils was significantly increased (p < 0.0005 and p < 0.006, respectively).nnnCONCLUSIONnInterleukin-8 can induce cervical ripening in nonpregnant and pregnant rabbits.

Stretching of fetal membranes increases the concentration of interleukin-8 and collagenase activity.

OBJECTIVEnThe aim of this study was to determine whether stretching of fetal membranes can increase interleukin-8 concentration and collagenase activity.nnnSTUDY DESIGNnStrips of whole fetal membranes, amnion, or muscles of the lower uterine segment were stretched for 2 or 4 hours. Interleukin-8 and collagenase activity were measured in homogenized control and stretched samples. Immunohistochemical staining for interleukin-8 was carried out.nnnRESULTSnThe interleukin-8 concentration increased significantly after the whole fetal membranes were stretched for 2 and 4 hours (p <0.0007 and 0.001, respectively). Also, stretching of amnion and muscles of the lower uterine segment for 2 and 4 hours increased the concentration of interleukin-8 significantly (p <0.0007 after 2 and 4 hours, respectively). Collagenase activity was significantly increased after stretching of amnion, amniochorion, and muscles of the lower uterine segment for 4 hours (p <0.0007, 0.006, and 0.0007, respectively). After stretching, samples were darkly stained for interleukin-8 compared with control nonstretched samples.nnnCONCLUSIONnStretching of amnion, amniochorion, and muscles of the lower uterine segment increased interleukin-8 production and collagenase activity.

Plasma P selectin (GMP-140) and glycocalicin are elevated in preeclampsia and eclampsia: Their significances ☆ ☆☆ ★

OBJECTIVEnWe measured the concentrations of plasma P selectin (or GMP-140) and glycocalicin in preeclamptic and eclamptic women. Correlations between these two parameters and blood pressures, platelet counts, or plasma thrombin-antithrombin complex values were evaluated.nnnSTUDY DESIGNnBy use of enzyme-linked immunosorbent assays we measured the plasma GMP-140 and glycocalicin levels in normal pregnancies (n = 10) and preeclamptic (n = 10) and eclamptic (n = 20) pregnancies. The glycocalicin index was calculated as follows: (glycocalicin x [250 x 10(6)/ml])/(Individual platelet counts). Correlations between plasma GMP-140, glycocalicin, glycocalicin index values, blood pressures, platelet counts, and plasma thrombin-antithrombin complex values were analyzed.nnnRESULTSnPlasma GMP-140 levels were found to be significantly elevated in preeclamptic (p < 0.0005) and eclamptic cases (p < 0.0001) compared with normotensive controls. Plasma glycocalicin (p = 0.01, 0.007) and glycocalicin index (p = 0.005, 0.002) values were also markedly elevated in preeclamptic and eclamptic patients compared with normal pregnant patients. Significant correlations between platelet counts or plasma thrombin-antithrombin complex levels and their corresponding plasma GMP-140 and glycocalicin and glycocalicin index values have been found in preeclamptic and eclamptic cases. However, blood pressures had correlations with GMP-140, glycocalicin, and glycocalicin index values in eclamptic cases.nnnCONCLUSIONSnWe demonstrated an elevation of plasma GMP-140 and platelet glycocalicin in preeclampsia and eclampsia. This study also reflects the usefulness of glycocalicin as a marker of platelet activation or turnover and endothelial dysfunction in these diseases.

Activated neutrophil by endothelin-1 caused tissue damage in human umbilical cord

Immunostaining of human neutrophils incubated with endothelin-1 (ET-1) showed intense and spreading pattern of anti human granulocyte elastase within the cytosol. That reflected neutrophil activation followed by the release of granule contents by ET-1. In contrast, PBS (phosphate buffered saline) treated neutrophils showed localized and faintly stained granules. Intracellular calcium in fura-2 loaded neutrophils was measured at 340/380 nm. A dose and time dependent increase in intracellular calcium by ET-1 occurred in human single neutrophil. Elastase activity assay was done with chromogenic substrate S2484. ET-1 induces dose and time dependent increase in elastase activity in neutrophil suspensions like ionophore A23187. A similar time dependent increase in elastase activity was retained even after repeated wash and ET-1 treatment. That confirmed the viability of most of the neutrophils after each treatment. In umbilical cord preparations, ET-1 treated neutrophils could migrate from the venous lumen into the tissue matrix of the umbilical cords. Hematoxylin and eosin staining revealed a massive tissue destruction in ET-1 activated neutrophil treated cords when compared to sham control and untreated neutrophil injected cords. Immunostaining with monoclonal anti human elastase revealed an intense staining in former sections when compared to the others. We suggest that ET-1 activated neutrophil might play a major role in endothelial injury and tissue damage in conditions with high blood level of endothelin.

Coagulation in vivo microcirculation and in vitro caused by endothelin-1

This study was designed to elucidate the participation of endothelin-1(ET-1) in vivo and in vitro coagulation. The microvascular hemodynamic changes in terms of intravascular thrombus formation in rat mesentery induced by the superfusion of ET-1 (0.5, 1 and 2 pmol) were visualized by an intravital microscope system assisted by television-video tape recorder system. In addition to vasoconstriction we observed the blockade of circulation by clumps resembling thrombus in a dose dependent fashion by ET-1. Thrombus formation could be attenuated by pretreatment with superfusion of 3.8% Na citrate solution but not by the prior superfusion of 1 to 3 ng of nitroglycerine. Thrombus formation was found after the administration of 10 microliters of CaCl2 (100 nM) solution in Na citrate (3.8%, 20 microliters) and ET-1 treated field. In vitro study, a dose dependent increase in TAT (thrombin-antithrombin complexes) and decrease in AT III (antithrombin III) (%) activity, the prolongation of PT (prothrombin time) and APTT (activated partial thromboplastin time) was found by administering ET-1 immediately in native (unanticoagulated) blood in silicon coated test tubes (p < 0.05; n = 6). However in citrated blood, TAT complexes, AT III (%) activity, PT and APTT were not significantly changed after administration of the same doses of ET-1 (p > 0.05; n = 6). Therefore, this study suggested that endothelin-1 caused intravascular thrombosis and enhanced intra test tube coagulation which could be attenuated by blocking ionic calcium.

Mechanical stretching induces interleukin-8 gene expression in fetal membranes: a possible role for the initiation of human parturition

OBJECTIVEnInterleukin-8 (IL-8) is known to play a crucial role in human parturition. We aimed to study the effect of mechanical stretching on the expression of IL-8 in fetal membranes (amniochorion) and decidua.nnnSTUDY DESIGNnWe examined the expression of IL-8 and its receptor in fetal membranes (amniochorion) and decidua by immunohistochemical staining. Also, we studied the synthesis of IL-8 messenger RNA (mRNA) in the fetal membranes before and after stretching.nnnRESULTSnWe found that mechanical stretching within physiological limit increased IL-8 messenger RNA (mRNA) synthesis in fetal membranes and decidua in a time- and load-dependent manner. Application of mechanical force led to markedly increased staining of IL-8 receptor in decidual cells but not in amnion or chorion cells.nnnCONCLUSIONnThese results suggested that mechanical stretching was a candidate for one of the signals important for production of IL-8 in fetal membranes and decidua and probably for initiation of a cytokine network at amniochorio-decidual interface through increased expression of IL-8 receptors.

Urinary trypsin inhibitor prevents uterine muscle contraction by inhibition of Ca++ influx.

OBJECTIVEnThe aim of this research was to elucidate the mechanism of action of urinary trypsin inhibitor, a Kunitz-type protease inhibitor, in suppressing uterine muscle contraction.nnnSTUDY DESIGNnAn isometric uterine contraction test was used to study this inhibitory effect of urinary trypsin inhibitor on the myometrium. Oxytocin, prostaglandin F2 alpha, and lipopolysaccharide were used to stimulate myometrial contraction. Prostaglandins F2 alpha and E2 were measured in the buffer solution. Influx of calcium into uterine smooth muscle cells was assessed by digital imaging microscopy.nnnRESULTSnAfter incubation with urinary trypsin inhibitor or fetal urine, myometrial contractions stimulated by oxytocin, prostaglandin F2 alpha or lipopolysaccharide were suppressed completely. The concentrations of prostaglandins F2 alpha and E2 in the buffer solution during the isometric contraction test were significantly increased by lipopolysaccharide stimulation, but when urinary trypsin inhibitor was present in the buffer solution the concentrations of prostaglandins F2 alpha or E2 did not change significantly. Preincubation with urinary trypsin inhibitor also inhibited calcium influx, resulting in no detectable change in the intracellular free calcium concentration of smooth muscle cells.nnnCONCLUSIONnWe proposed that urinary trypsin inhibitor from fetal urine inhibits uterine muscle contraction by regulation of intracellular Ca++.

Endothelin-1 increased immunoreactive von Willebrand factor in endothelial cells and induced micro thrombosis in rats

This study was performed (i) to investigate the interaction between ET-1 and endothelial cells and (ii) to study the role of ET-1 in in vivo thrombosis. Fura-2AM loaded human umbilical endothelial cell cultures were incubated with 0, 25, 50 and 100 pmol of ET-1 for 24 hours (n = 6) at 37 degrees C. Fura-2 released in the media was measured by spectroflurophotometer at wavelength of 350 nm excitation and 500 nm emission. We found significant (p < 0.01) and dose dependent decrease in Fura-2 release by the cells indicating increased intracellular calcium in HUVEC. Increased calcium by ET-1 was also confirmed at single cell level by fluorescence digital image analysis using Fura-2AM. 5 ml solution of ET-1 (100 pmol/ml) was injected within the venous lumen of umbilical cords (of normal pregnancy) clumped at both ends and incubated at a temperature 37 degrees C for 3 hours (n = 7). We found intensely stained immunoreactive von Willebrand factor (vWF) on the endothelial cells of ET treated umbilical cords when compared with sham control (Umbilical cords incubated with phosphate buffer saline; n = 7). Intravenous ET-1 infusions at a rate of 1 nmol/kg/hour for 2 hours (cases, n = 7) and 5% dextrose infusions (sham control, n = 7) were performed in rats. Aorta, kidney and liver tissues were obtained to perform immunostaining with polyclonal antibody to vWF and fibrinogen. ET-1 treated rat tissues showed intense staining for vWF and fibrinogen intravascularly at hte same site.(ABSTRACT TRUNCATED AT 250 WORDS)

HELLP Syndrome-Like Biochemical Parameters Obtained with Endothelin-1 Injections in Rabbits

We studied the effects of bolus injections and infusion of endothelin-1 (ET-1) in female rabbits by measuring serum glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), lactic dehydrogenase (LDH), antithrombin III (AT-III), thrombin antithrombin (TAT) complexes, platelet counts and indirect bilirubin. Two successive bolus doses of 0.125 and 0.25 nmol/kg of ET-1 with an interval of 30 min were given to conscious non-pregnant female rabbits (n = 8). GOT, GPT and LDH were found to be significantly increased after injections of ET-1 (p < 0.02, p < 0.04 and p < 0.05, respectively). The percent AT-III activity decreased significantly (p < 0.005). Vasospasm of the hepatic artery was demonstrated by angiography with the same bolus doses in rabbits. There was also an increase in GOT (p < 0.003), GTP (p < 0.05), LDH (p < 0.007), indirect bilirubin (p > 0.07) and TAT complexes (p < 0.02) and a decrease in AT-III (p < 0.03) and platelet counts (p < 0.02) in rabbits (n = 10) after 24 h of continuous infusion of ET-1 (0.6 nmol/kg/h). Histological examination of rabbit liver tissues showed varying degrees of ischemic necrosis of hepatocytes. Thus this study suggests that exogenously administered ET-1 causes vasospasm and liver ischemia resulting in HELLP syndrome-like blood parameters in rabbits.

Urinary Trypsin Inhibitor Has a Protective Effect on the Amnion

Urinary trypsin inhibitor-related substance (UTI-R) was measured in the amniotic fluid (n = 30), first neonatal urine (n = 10), meconium (n = 10), and adult urine (n = 10). The concentration of UTI-R excreted in the neonatal urine was significantly higher than in adult urine and meconium. Positive immunostaining for UTI was observed in amnion, umbilical cord and placenta. The effect of UTI was studied on the amnion using fura-2-acetoxymethyl ester. Interleukin-1 beta (IL-1 beta) and tumor necrosis factor-beta (TNF-beta) led to a significant increase in relative fluorescence release (RFR) of fura-2 (p < 0.05 and p < 0.02, respectively) while endotoxin did not show significant changes of RFR of fura-2. The effect of IL-1 beta and TNF-beta was almost abolished after incubation of UTI with the amnion cells (p < 0.03). This suggests that UTI may have a protective effect on the amnion especially against IL-1 beta and TNF-beta.