Preliminary trials of a specific gravity technique in the determination of early embryo growth potential†.

STUDY QUESTION: Can a modified specific gravity technique be used to distinguish viable from nonviable embryos? SUMMARY ANSWER: Preliminary data suggests a modified specific gravity technique can be used to determine embryo viability and potential for future development. WHAT IS KNOWN ALREADY: Single embryo transfer (SET) is fast becoming the standard of practice. However, there is currently no reliable method to ensure development of the embryo transferred. STUDY DESIGN, SIZE, DURATION: A preliminary, animal-based in vitro study of specific gravity as a predictor of embryo development using a mouse model. PARTICIPANTS/MATERIALS, SETTING, METHODS: After a brief study to demonstrate embryo recovery, experiments were conducted to assess the ability of the specific gravity system (SGS) to distinguish between viable and nonviable embryos. In the first study, 1-cell mouse embryos were exposed to the SGS with or without previous exposure to an extreme heat source (60°C); measurements were repeated daily for 5 days. In the second experiment, larger pools of 1-cell embryos were either placed directly in culture or passed through the SGS and then placed in culture and monitored for 4 days. MAIN RESULTS AND THE ROLE OF CHANCE: In the first experiment, viable embryos demonstrated a predictable pattern of descent time over the first 48 h of development (similar to previous experience with the SGS), while embryos that were heat killed demonstrated significantly altered drop patterns (P < 0.001); first descending faster. In the second experiment, average descent times were different for embryos that stalled early versus those that developed to blastocyst (P < 0.001). Interestingly, more embryos dropped through the SGS developed to blastocyst than the culture control (P < 0.01). LIMITATIONS, REASONS FOR CAUTION: As this is a preliminary report of the SGS technology determining viability, a larger embryo population will be needed. Further, the current in vitro study will need to be followed by fecundity studies prior to application to a human population. WIDER IMPLICATIONS OF THE FINDINGS: If proven, the SGS would provide a noninvasive means of assessing embryos prior to transfer after assisted reproductive technologies procedures, thereby improving fecundity and allowing more reliable SET. STUDY FUNDING/COMPETING INTERESTS: The authors gratefully acknowledge the funding support of the U.S. Jersey Association, the Laura W. Bush Institute for Women's Health and a Howard Hughes Medical Institute grant through the Undergraduate Science Education Program to Texas Tech University. None of the authors have any conflict of interest regarding this work. TRIAL REGISTRATION NUMBER: none.

Increases in progesterone after human chorionic gonadotropin administration may predict cycle outcome in patients undergoing in vitro fertilization-embryo transfer.

In this study, both IVF-ET cycle and pregnancy outcome were compared with increases seen in P concentration during the 24-hour period from 12 hours before to 12 hours after hCG administration. A significantly higher PR (P < 0.001) was seen in women who had at least a threefold increase in P during this time period. Further, no pregnancies were reported from women with a less than twofold increase in P.

Seminal concentssrations of total and ionized calcium from men with normal and decreased motility.

In this study, sperm motility, velocity, and progression were compared with the total and Ca++ concentrations in the SF from men with normal and decreased motility (less than 60%). No significant difference in SF total calcium content was observed in men with normal and hypomotility. However, a statistically significant decrease in seminal Ca++ was observed in those men with decreased motility, when compared with that of men with normal motility.

Regulation of DNA synthesis in Leydig cells obtained from neonatal pig testes.

Three distinct waves of Leydig cell development are found in the pig testes, which occur during fetal, perinatal, and prepubertal periods. Proliferation of Leydig cells is primarily regulated by luteinizing hormone (LH); however, effects of LH on proliferation of immature rat Leydig cells are mediated by specific growth factors and cytokines such as transforming growth factor-alpha (TGFalpha), insulin-like growth factor-1 (IGF-1), interleukin-1beta (IL-1beta), steroidogenesis-inducing protein (SIP), and TGFbeta. The objective of the present study was to identify growth factors that could possibly be involved in the proliferation of Leydig cells in the neonatal pig testis. Leydig cells were isolated from 3- to 5-d-old pig testes, cultured for 48 hr in serum-free media, washed, and treated with hCG and/or IGF-1, epidermal growth factor (EGF), IL-1beta, SIP, and TGFbeta for 18 hr. Tritiated thymidine incorporation into DNA was assessed over a subsequent 4-hr period. Incorporation of [3H]-thymidine was stimulated by hCG treatment with a 2.3-fold increase over control cultures. SIP also induced a significant increase (P < 0.0001) in the incorporation of [3H]thymidine into Leydig cell DNA. Similarly, EGF and IGF-1 also increased DNA synthesis in neonatal porcine Leydig cells, whereas IL-1beta had no effect. TGFbeta had very little, if any, effect on DNA synthesis when added alone, but inhibited the stimulatory effects of other mitogens (SIP, hCG, EGF/TGFalpha, and IGF-1). Our results indicate that these growth factors may play a role in the LH/hCG-dependent proliferation of Leydig cells during the perinatal period of development.

Evidence for the direct inhibition of endothelin-1 secretion by hemoglobin in human endothelial cells.

The actual hemoglobin (Hb) contribution to endothelin-1 (ET-1) production in human umbilical vein endothelial cells (EC) was investigated. Cells were incubated with 0.1 mmol or 0.3 mmol of bovine: 1) unmodified (U) ferrous-Hb; 2) U-ferric-Hb; 3) U-ferryl-Hb; 4) polymerized low molecular weight (m.w.) Hb with chemically modified surface (< 400 kDa); and 5) glutaraldehyde polymerized, high m.w. Hb (< 1020 kDa). The incubation medium was tested at 6 and 24 hr for lactate dehydrogenase (index of cellular injury), and for ET-1 release by the cells. Before radioimmunoassay, the ET-1 was extracted from cell culture medium by a two-step purification procedure: 1) ultrafiltration, and 2) column extraction with C18 cartridges. The data suggested that the oxidation status of Hb and its concentration play an important role in causing EC injury. The highest toxicity was observed when EC were incubated with 0.1 mmol of ferryl-Hb, and there was no toxicity with 0.3 mmol of ferric-Hb. These results indicate that the ferric-Hb and low m.w. polymerized Hb at a concentration of 0.1 mmol did not alter ET-1 synthesis and produced a level similar to that of the control. However, it was found that ferryl-Hb and ferrous-Hb in a concentration of 0.1 mmol significantly reduced ET-1 release. All Hbs at a concentration of 0.3 mmol markedly inhibited the production of ET-1. The greatest decrease in ET-1 levels was produced by ferryl-Hb, and the lowest by ferric-Hb and low m.w. polymerized Hb. The Hb's inhibitory effect was more pronounced at 24 hr of incubation. It was also found that although Hb molecules showed a high degree of cross-reactivity with polyclonal anti ET-1 antibodies, the presence of different Hb solutions in the EC culture medium did not change the immunologic properties of ET-1 peptide. In conclusion, Hb inhibitory activity toward ET-1 production might be related to Hb mediated endothelial oxidative injury.

Improved blood substitute: evaluation of its effects on human endothelial cells.

The authors have previously documented that appropriate chemical and pharmacologic modification of the hemoglobin molecule are required to attenuate certain pathophysiologic reactions of the reticuloendothelium. The current study further investigates the molecular responses of human coronary artery endothelial cells to a high concentration (0.4 mmol) of 1) unmodified bovine hemoglobin; and 2) an improved blood substitute that comprises hemoglobin cross-linked intramolecularly with o-adenosine triphosphate and intermolecularly with o-adenosine, and conjugated with reduced glutathione. In this study, the scavenging effect of hemoglobins toward nitric oxide (NO) was evaluated by the measurement of nitrite (NO2-) and nitrate (NO3-) formation. The pro-oxidant effect of hemoglobin on endothelial cells was examined by the measurement of intracellular reduced glutathione, and by monitoring the formation of lipid hydroperoxides and 8-iso prostaglandin F2alpha, a novel potent vasoconstrictor, which is produced by a noncyclooxygenase mechanism involving free radical catalyzed peroxidation of arachidonic acid. The inflammatory reactions of endothelial cells were evaluated by the expression of the adhesion molecule, intracellular adhesion molecule-1, and the activation of nuclear transcription factor, nuclear factor kappaB. In additional, endothelial cell responses were investigated by analysis of intracellular ionized calcium concentrations. Results indicate that unmodified hemoglobin in a concentration of 0.4 mmol/L can aggravate endothelial cell oxidative and inflammatory responses. This hemoglobin produced a significant (p < 0.01) depletion of reduced glutathione, acceleration of lipid peroxidation, and a greater influx of Ca2+. The formation of 8-iso prostaglandin F2alpha increased compared with the control cells (p < 0.01). Unmodified hemoglobin was found to be a potent scavenger of NO, great activator of nuclear factor kappaB, and a stimulator of intracellular adhesion molecule-1 expression. Contrarily, the improved blood substitute did not appear to induce oxidative stress nor to increase the intracellular Ca2+. The concentration of 8-iso prostaglandin F2alpha was similar to that in the control cells, whereas the formation of NO2-/NO3- was much lower (p < 0.05) than in the unmodified hemoglobin group. The effect of an improved blood substitute can be linked with the anti-inflammatory and cytoprotective properties of adenosine, which is used as a cross-linker and surface modifier, and the type of the chemical modification procedure that lowers hemoglobin pro-oxidant potential.

The dosage of human chorionic gonadotropins influences the relationship between progesterone and cycle outcome during in vitro fertilization-embryo transfer.

OBJECTIVE: To determine if the dose of hCG affects the initial rise in progesterone seen in patients undergoing IVF-ET and therefore affects it usefulness as a predictor of cycle outcome. DESIGN: Comparison of the rise in progesterone with cycle outcomes for IVF patients receiving 5,000 or 10,000 mIU hCG to stimulate oocyte maturation. SETTING: University-based infertility program. Patients-One hundred six patients undergoing IVF-ET on a long protocol of down-regulation with GnRH, hMG stimulation, and hCG to stimulate oocyte maturation. Stimulation protocol varied only in dose of hCG [5,000 mIU (N = 72) vs. 10,000 mIU (N = 34)]. MAIN OUTCOME MEASURE(S): Rise in progesterone from 12 hours before to 12 hours after hCG administration and its relationship with cycle outcome. RESULTS: All 106 women exhibited a rise in progesterone following the administration of hCG. As seen in earlier studies, there appeared to be a relationship between minimal progesterone increases (<3-fold) and cycle failure in patients receiving 5,000 mIU (P < .02). However, using the criteria of the previous study, there appears to be no relationship between progesterone and cycle outcome in patients receiving 10,000 mIU (P = .30). Further, the higher dose of hCG appeared to induce greater increases in progesterone over the 24-hour period examined (P < .02). After readjustment of the critical value to 3.5-fold, there was an increased tendency toward cycle failure in women exhibiting a minimal progesterone increase. Unlike the relationship associated with 5,000 mIU hCG, though, the relationship between 10,000 mIU hCG and progesterone levels was not statistically different (P = .10). CONCLUSIONS: Increasing the dose of hCG used to stimulate oocyte maturity shifts the previously described relationship between progesterone and IVF-ET-cycle outcome. However, while it remains unclear if progesterone can be used as a predictor of outcome at the higher hCG dose, it appears clear that a relationship exists between minimal progesterone response to hCG and cycle failure.

Preliminary evaluation of a unique freezing technology for bovine spermatozoa cryopreservation.

Artificial insemination (AI) and semen cryopreservation has significantly improved the breeding potential of male animals. However, current freezing techniques commonly result in reduced semen quality. Ten years ago, a unique freezing technology (UFT) was developed for the freezing of foodstuffs and other materials. Previous work from this laboratory has demonstrated the UFT to be a superior method of freezing for a number of cell types. In a preliminary study, the UFT was compared with the conventional freezing methodology of bovine semen. Semen samples were collected from an angus (Bull A) and a gelbivich bull (Bull B), prepared using a conventional bovine cryoprotectant, and frozen in the UFT or in liquid nitrogen (LN) mist. The samples were stored in LN before being thawed and assessed for the semen parameters of motility and forward progression. Preliminary results suggest the UFT is equivalent to current techniques in the cryopreservation and recovery of bovine semen, and with modification, possibly a superior technique for semen freezing. Further studies using larger sample populations, and using a CASA system to evaluate motility, forward progression and linearity are merited.

Taurine binding by rat retinal membranes.

Retinal membrane preparations contain endogenous taurine which is difficult to remove. By repeatedly washing the membranes in buffer the taurine content was reduced from 11.7 +/- 2.5 to 2.5 +/- 0.5 nmol/mg protein. However, complete elimination of the endogenous taurine from the membrane preparation was not achieved. Binding of [3H]-taurine to rat retinal membrane preparations revealed both high and low affinity binding sites. A Hill coefficient of 0.71 suggested that cooperativity may be involved in the taurine binding process. Taurine binding was sodium dependent with maximum binding achieved at 118 mM. At suboptimal concentrations of sodium ions (30 mM) only one binding site was observed which appears to be the high affinity binding site. Analogues of taurine were tested for their effectiveness in displacing taurine from its binding site. Hypotaurine, beta-alanine, gamma-aminobutyric acid, and guanidinoethanesulfonic acid were the most potent displacers.

Estimation of fetal weight before and after amniotomy in the laboring gravid woman.

OBJECTIVE: This study was undertaken to search for differences between fetal weights estimated both ultrasonographically and clinically before and after amniotomy in laboring gravid women. STUDY DESIGN: Estimates of fetal weight (ultrasonographic and clinical) were obtained for laboring gravid women before and after amniotomy. These estimates were compared with actual birth weights determined post partum. RESULTS: One hundred sixty-two patients completed the study protocol. Comparisons made with unpaired Student t test analyses demonstrated a difference (P <.001) between ultrasonographically estimated fetal weights before and after amniotomy. Simple regression analysis showed a correlation between both ultrasonographic and clinical estimates of fetal weight and actual birth weights before and after amniotomy, with postamniotomy clinical estimates having the strongest correlation (ultrasonographic preamniotomy estimate, R = 0.717; ultrasonographic postamniotomy estimate, R = 0.630; clinical preamniotomy estimate, R = 0.742; and clinical postamniotomy estimate, R = 0.788). Of all ultrasonographic parameters measured, preamniotomy abdominal circumference correlated best with actual birth weight (R = 0.730). CONCLUSION: Clinical estimates of fetal weight after amniotomy correlated well with actual birth weights. Preamniotomy abdominal circumference was the ultrasonographic parameter best for prediction of actual birth weight. Maternal weight affected clinical but not ultrasonographic estimates of fetal weight in this study. However, clinical estimates of fetal weight were actually superior to ultrasonographic estimates of fetal weight in this study.

Hormone release from cultured luteinized-granulosa cells mimics differences seen in vivo in patients undergoing IVF-ET.

OBJECTIVES: Previous research from this laboratory has suggested that a relationship exists between the increase in circulating progesterone concentrations at the time of hCG administration and cycle outcome in patients undergoing IVF. Progesterone (P) increases of threefold or better within the 24-h period surrounding hCG administration appeared to be associated with a higher pregnancy rate. These data suggest a functional difference in the luteinized-granulosa of patients undergoing IVF. To test this hypothesis: DESIGN: A split-split plot arrangement of treatments with two cell sources under five hormonal stimulations at four time points. METHODS: Luteinized-granulosa cells (LGC) were collected from patients with either a normal increase (> or = threefold = NC) in circulating P (n = 4) or those with lower P increases (AC; n = 4). The cells were isolated by Ficoll gradient centrifugation and then cultured in 24-well culture plates using a modified media 199 containing 100 mIU/ml of hMG, FSH, LH, hCG, or a nonhormonal control to stimulate steroid-hormone production. At time points 2, 4, 6, and 8 days, media from each well was frozen for later hormone assay and replaced with fresh media containing the same stimulating factor. After all the media had been collected, P and estradiol (E2) released into the media were measured using radioimmunoassay. RESULTS: Results indicate a higher media concentration of P (P < 0.001), but not E2 (P = 0.254), from NC, regardless of hormone stimulation or time in culture, when compared to the media from AC. Media concentrations of E2 were affected by a cell source by hormone stimulation by time interaction (P < 0.001) with varying effects. Media from NC maintained a constant E2 of between 1000-3000 pg/ml over the 8-day period (P = 0.163). However, media from AC demonstrated a stimuli-dependent E2 release (P < 0.001) ranging from < 1000 to over 11,000 pg/ml. CONCLUSIONS: These data appear to support the existence of functionally different populations of luteinized-granulosa cells from patients undergoing IVF-ET.

Adhesion-promoting properties of dyes routinely used during fertility surgeries.

PURPOSE: With the link between peritoneal adhesions and infertility well established, it is critical that materials used in pelvic surgery be tested for their adhesion-forming properties. The current study examined the adhesion-inducing properties of two dyes routinely used for visualization during pelvic surgery. DESIGN: In vivo and in vitro examination of the effects of the dyes methylene blue and indigo carmine on adhesion formation in a mouse model. METHOD: A series of three experiments was conducted. In the first, dyes were injected directly into the peritoneal cavity. The mice were then sacrificed at one of two time points and the peritoneal cavity examined for adhesion formation. In addition, because of their purposed role in adhesion formation, macrophages from the cavity were examined for signs of dye-induced activation. Further studies of macrophage activation were then conducted in vitro to determine the effects of dye concentration and exposure time on the activation process. RESULTS: Both methylene blue and indigo carmine appeared to induce adhesion formation as well as macrophage activation in vivo. Further, long-term exposure to visual concentrations of both dyes appeared to induce macrophage activation. However, only those macrophages exposed to methylene blue exhibited signs of activation when the exposure time was limited to times equivalent to those which might be expected during surgery. CONCLUSION: Of the two dyes tested, indigo carmine might be the dye of choice in surgeries where fertility is to be maintained.

Preliminary trial: motility comparisons of a unique freezing technology (UFT) to liquid nitrogen mist methodology for cryopreservation of porcine spermatozoa.

The motility outcomes of boar semen frozen with newly developed freezing techniques using a new unique freezing technology (UFT) compared with traditional liquid nitrogen methodology were investigated with the intent of improving current fertility outcomes using semen. The UFT is an electronically controlled cooling chamber that houses an organic fluid bath that can be maintained at temperatures below 0 degrees C without solidifying to freeze samples. Four ejaculates from four different boars were collected for this trial. Samples were handled consistently during the pre- and post-freeze processing. From each ejaculate, samples were separated into eight cryopreservation treatment groups, six UFT variations and two control liquid nitrogen groups, immediately before freezing, in replicates of two. After the initial cryopreservation was complete, all samples were stored in liquid nitrogen for at least 48 h. Post-thaw motilities and original motility return percentages were assessed on a random, individual-sample basis. After the initial evaluations, samples from two boars were recollected and frozen using the UFT for breeding purposes. Four sows were bred with the UFT frozen semen to confirm fertility capability. When assessing the individual UFT techniques, all of six UFT techniques had improved post-thaw motilities. However, treatments F (micro = 29%, return micro = 37%) and J (micro = 27%, return micro = 34%) showed the highest statistical improvement for post-thaw (p < 0.05) and original motility percent returns (p < 0.05) when compared with either the control cryo-tube (micro = 15%, return micro = 19%) or straw groups (micro = 12%, return micro = 16%). The UFT semen had a 50% conception rate, with an average of seven piglets from the sows that farrowed. Our preliminary data suggest a higher motility return with a slower pre-freeze phase below the freezing point before the acceleration to liquid nitrogen temperatures. The preliminary data suggest that the UFT could be utilized as a potential cryopreservation option for boar semen.

Modified hemoglobin solution, with desired pharmacological properties, does not activate nuclear transcription factor NF-kappa B in human vascular endothelial cells.

The aim of the present study was to evaluate the role of hemoglobin (Hb) and the contribution of chemically modified Hb solutions on the activation of nuclear transcription factor. NF-kappa B, and propagation of oxidative stress within human vascular endothelial cells. The activation of an oxidative stress-sensitive NF-kappa B can be linked with the propagation of an inflammatory state via rapid induction of genes for several pro-inflammatory mediators. Human coronary artery endothelial cells (HCAEC) were cultured on glass coverslips or cell culture plates to confluence. Then, the cells were incubated for up to 18 hours with endothelial basal medium (EBM) supplemented with 5% FBS and test agents in a concentration of 0.1 and 0.2 mmol: 1) unmodified bovine Hb (UHb): 2) modified Hb solution polymerized with glutaraldehyde (GLUT-Hb), and 3) a novel modified Hb solution (Hb-PP-GSH) prepared according to our patented procedure (U.S. Patent No. 5,439,882). The positive control for the NF-kappa B activation study included a treatment of the cells with: I) endotoxin: IL-1; TNF; and H2O2. Results indicate that Hb's pro-oxidant potential was influenced by the type of chemical modification procedure. The GLUT-Hb autoxidation rate, peroxidase-like activity and reactivity with H2O2/ferryl species formation were higher as compared to UHb, by 15%, 35% and 30%, respectively. However, pro-oxidant potential of Hb-PP-GSH was significantly lower than that of UHb (by 22%, 12% and 28%, respectively). The extent of oxidative stress of the HCAECs was found to be the Hb modification-type and concentration dependent. Although the highest endothelial lipid peroxidation and the largest depletion of intracellular GSH was associated with 0.2 mmol of GLUT-Hb, the Hb-PP-GSH did not produce significant changes when compared to the control cells. The UHb generated a moderate oxidative stress to the endothelium. The immunofluorescent and EMSA results indicate a correlation between the type of Hb chemical modification and the induction of NF-kappa B nuclear translocation. We found that GLUT-Hb rapidly activated NF-kappa B and induced nuclear translocation. Treatment of the cells with an increasing amount of UHb leads to the partial nuclear induction of NF-kappa B. However, Hb-PP-GSH did not activate NF-kappa B directly. In this study, the positive control cells treated with endotoxin, IL-1 or TNF demonstrated full nuclear translocations, whereas H2O2 caused only partial induction. In conclusion, nuclear translocation of NF-kappa B by Hb solutions might be dependent on Hb's pro-oxidant potential and extent of Hb-mediated endothelial oxidative stress. Besides the low oxidative potential of Hb-PP-GSH, the observed lack of NF-kappa B activation by this Hb solution can be also related to the anti-inflammatory properties of adenosine which is used in our novel modification procedure. In this study, only the Hb-PP-GSH, cross-linked intramolecularly with o-adenosine triphosphate and intermolecularly with o-adenosine, and combined with reduced glutathiore, was shown to be non-toxic to the endothelium and promises to be an effective free-Hb based blood substitute.

Protective effect of selenium on hemoglobin mediated lipid peroxidation in vivo.

The toxicity of hemoglobin (Hb) solutions is related, at least in part, to the generation of oxygen free radicals with consequent induction of lipid peroxidation. The present study was designed to examine whether selenium (Se) may prevent the oxidative damage observed after Hb administration. Three groups of rats were compared; (I) the negative control group receiving autotransfusion; (II) the positive control group with replacement of 40% total blood volume (TBV) with modified bovine Hb solution; and (III) the experimental group which received dietary supplemented selenium (Na2SeO3) in daily doses of 5 micrograms.kg body wt-1 in drinking water, 4 days before and 3 days after administration of Hb solution in the same volume as in group II. Three days after Hb injection, all animals were sacrificed. Oxidative stress was determined by measuring conjugated dienes (CD) and thiobarbituric acid reactants (MDA) in homogenates of the perfused liver, heart, lungs, kidney, brain and plasma. Additionally, the 45k x g supernatants of the organs homogenates and plasma were assayed for the antioxidant enzymes activity: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and the intracellular level of reduced glutathione (GSH). Also, a measurement of nonprotein bound intracellular free iron (Fe) and tissue Se concentrations was performed. Simultaneously, injury dysfunction of vital organs was assessed by the measurement of plasma LDH, SGPT, creatinine, blood PaO2 and by histopathological studies. Results indicate that the exchange transfusion with Hb solution introduced significant increases in CD and MDA formation, particularly in the liver and heart tissues, and in plasma. While the values of the SOD and CAT in the liver and heart tissue were generally altered, the SOD/CAT ratio was also increased. After the Hb injection, activity of GSH-Px remained unchanged and was associated with significant depletion of GSH. The plasma levels of SGPT and LDH were increased, but the creatinine and PaO2 was similar to that of the control and corresponded with histopathological findings. The liver and heart intracellular free Fe was found to be higher than that of control. Treatment with Se was very effective in the prevention of oxidative damage introduced by Hb. Full protection from MDA formation was noted in liver tissue (p < 0.001). Also, plasma levels of MDA, SGPT and LDH were significantly decreased and appeared similar to that of the control group (I). Treatment with Se increased liver (p < 0.05) and plasma (p < 0.1) level of GSH-Px.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of adhesion molecules and von Willebrand factor in human coronary artery endothelial cells incubated with differently modified hemoglobin solutions.

Previous studies have established a linkage between free Hb molecules and the production of inflammatory mediators by the reticuloendothelial cells. An important aspect of the endothelial response to the inflammatory stimuli is the expression of adhesion molecules on the luminal surface. Therefore, the present study was designed to investigate the effects of various free-Hb based oxygen carrying solutions on the intracellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule-1 (VCAM-1) and also von Willebrand factor (vWF) expression by human endothelium. Human coronary artery endothelial cells (HCAEC) were cultured on glass coverslips until they reached confluence, then incubated for 18 hours with endothelial basal medium (EBM) supplemented with 5% FBS and a 0.1 mmol or 0.2 mmol of the bovine Hb solutions: 1) pure unmodified bovine Hb (UHb); 2) modified bovine Hb solution (Hb-PP-GSH) prepared according to our newly developed procedure (U.S. Patent No. 5,439,882); and 3) modified bovine Hb solution polymerized with glutaraldehyde (GLUT-Hb). The HCAECs were also incubated with EBM (negative control) and EBM containing bacterial endotoxins in a concentration of 50 EU/ml (positive control). After treatment, cells were exposed to primary antibodies; anti-human ICAM-1, anti-human VCAM-1 or anti-human vWF, and consequently to the secondary antibody (fluorescein isothiocyanate-conjugated F(ab)2). Immunofluorescence analysis revealed different expressions of ICAM-1 and VCAM-1 on the surface membranes of variously treated cells. Although negative control cells had an undetectable level of adhesion molecules, the positive control cells, activated by endotoxin, exhibited high immunoreactivity for ICAM-1 and VCAM-1. The Hb's treated cells demonstrated differing degrees of activation. An insignificant expression of ICAM-1 was observed in HCAEC, following treatment with a 0.1 or 0.2 mmol of Hb-PP-GSH and 0.1 mmol of UHb. Cell treated with 0.2 mmol of UHb and both concentrations of GLUT-Hb demonstrated a massive expression of this adhesion molecule. A similar effects was observed during induction of VCAM-1. While a lack of expression was noted with both concentrations of Hb-PP-GSH and 0.1 mmol of UHb, the GLUT-Hb stimulated significant VCAM-1 induction at all tested concentrations. Immunofluorescence analysis confirmed the expression of vWF uniformly in HCAEC from the different experimental groups. The data suggest, vWF expression was unaffected by all but the GLUT-Hb treatment. In conclusion, the Hb stimulatory activity toward ICAM-1 and VCAM-1 inductions were related with the type of Hb chemical modification method. Although modification of Hb with glutaraldehyde potentiates adhesion molecules expression, our novel Hb modification procedure, which comprises intramolecular cross-linking with o-adenosine triphosphate and intermolecular with o-adenosine, and combined with reduced glutathione, apparently prevents these inflammatory events.