Nitric oxide generation affects pro- and anti-angiogenic growth factor expression in primary human trophoblast.

OBJECTIVES: Preeclampsia is associated with reduced trophoblast placenta growth factor (PGF) expression, elevated soluble fms-like tyrosine kinase-1 (sFlt-1) and decreased bioactivity of nitric oxide (NO). Elevated sFlt-1 reduces bio-availability of PGF and vascular endothelial growth factor (VEGF) leading to maternal endothelial dysfunction. Although NO can regulate gene expression, its ability to regulate trophoblast expression of angiogenic growth factors is not known. STUDY DESIGN: Human primary term trophoblast and JEG-3 choriocarcinoma cells were cultured under 21%O(2) or 1%O(2) conditions in the presence or absence of NO donor (SNP) or inhibitor (L-NAME). Effects on PGF, VEGF and Flt-1 isoform mRNA expression were determined by quantitative real-time PCR. Changes in expression of soluble protein isoforms of FLT-1 was monitored by ELISA. RESULTS: Hypoxia decreased PGF mRNA but increased VEGF, sFlt-1 and Flt-1 mRNA expression in trophoblast. Generation of NO in trophoblast under 1%O(2) culture conditions significantly reversed sFlt-1 mRNA and protein expression, independent of mFlt-1. Conversely NO generation in hypoxic trophoblast increased VEGF and PGF mRNA expression. CONCLUSIONS: NO production in primary human trophoblast cultures had divergent effects on pro-angiogenic (PGF, VEGF) versus anti-angiogenic (sFlt-1) mRNA expression, resulting in an enhanced pro-angiogenic gene expression environment in vitro.

Differential regulation of human PlGF gene expression in trophoblast and nontrophoblast cells by oxygen tension.

OBJECTIVE: To determine the mechanism for differential effects of low oxygen tension on human PlGF gene transcription in trophoblast and nontrophoblast cells. STUDY DESIGN: Human PlGF reporter clones and real-time RT-PCR were used to compare the effects of hypoxia on gene transcription in human trophoblast and nontrophoblast cell lines. Overexpression of HIF-1alpha, inhibition of HIF-1 function and biochemical assessments of HIF-1 co-factor interactions were used to characterize hypoxia response mechanisms regulating PlGF transcription. RESULTS: PlGF transcription is specifically inhibited by low oxygen tension in trophoblast but is induced in some nontrophoblast cells. Overexpression of HIF-1alpha in normoxic cells or inhibition of HIF-1 function in hypoxic cells did not significantly alter transcription patterns of the PlGF gene in either cell type. CONCLUSIONS: These results suggest that transcriptional repression of PlGF gene expression occurs in human trophoblast exposed to low oxygen tension but that PlGF transcription is stimulated in certain hypoxic nontrophoblast cells. However, regulation of PlGF transcription is not mediated by functional HIF-1 activity in either cell type.

Deferential regulation of placenta growth factor (PlGF)-mediated signal transduction in human primary term trophoblast and endothelial cells.

Increasing evidence supports that many common obstetrical complications may involve the disruption of normal placental and/or uterine vascular function. Placenta growth factor (PlGF) is an angiogenic factor that is abundantly expressed in the placenta, with primary site of synthesis being trophoblast. Receptors for PlGF include products of the fms-like tyrosine kinase (flt-1) gene which is expressed in several cell types including endothelial cells and trophoblast. PlGF activation of flt-1 in trophoblast induces the stress activated protein kinase (SAPK) signal transduction pathways, JNK (c-Jun-N-Terminal Kinase) and p38, with little induction of the extracellular signal-regulated protein kinase (ERK)-1/2 pathways. In contrast, PlGF induces strong ERK-1/2 activation, but little JNK or p38 responses in human umbilical vein endothelial cells (HUVEC). To better understand the biochemical functions of PlGF in trophoblast, we studied upstream signal regulatory molecules to determine those that are responsible for directing the divergent PlGF signal transduction responses in these cell types. PlGF induced similar activation of Nck and PLC-gamma in trophoblast and HUVEC. In marked contrast, SHP-2 and Gab2 were strongly activated by PlGF in endothelial cells but not trophoblast. These results suggest a general role for Nck and PLC-gamma in mediating PlGF signal transduction responses independent of the different downstream MAPK pathways activated. However, SHP-2 and Gab2 are regulatory molecules involved in the PlGF induction of different terminal pathways in HUVEC and trophoblast.

Low maternal serum levels of placenta growth factor as an antecedent of clinical preeclampsia.

OBJECTIVE: Maternal serum placenta growth factor levels have been shown to be significantly reduced in women with established preeclampsia. However, the temporal change in serum placenta growth factor levels before the clinical onset of preeclampsia is not known. STUDY DESIGN: Serum samples were collected from patients at the first prenatal (5-15 weeks' gestation), second-trimester (16-20 weeks' gestation), and third-trimester (26-30 weeks' gestation) visits. Serum placenta growth factor levels were determined and analyzed according to pregnancy outcome. RESULTS: Maternal placenta growth factor levels during normal gestation increased dramatically from the first to the third trimester. At the same gestational time points, in contrast, significantly lower serum placenta growth factor levels were found in patients in whom mild or severe preeclampsia eventually developed (P <.01). Low maternal serum placenta growth factor levels during early gestation were associated with a significant odds ratio for development of preeclampsia (P <.005). CONCLUSION: Relatively decreased levels of serum placenta growth factor occur before the onset of clinical preeclampsia, which suggests that placenta growth factor measurement could be used to discriminate those pregnancies predisposed to development of preeclampsia.

Increased vascular endothelial growth factor expression in human hearts with microvascular fibrin.

We have shown that microvascular changes that promote fibrin deposition in human cardiac allografts adversely affect clinical outcome. However, some allografts exhibit phenotypic changes in capillaries following the deposition of fibrin, which subsequently provide a significant survival advantage. The mechanism(s) involved in these capillary changes is(are) unknown. Similarly, although we have shown a significant temporal relationship between microvascular fibrin deposition and vascular endothelial growth factor (VEGF) immunoreactivity in cardiac allografts, the cellular source and relative changes in VEGF gene expression under these conditions are not known. Using immunocytochemical techniques, biopsies devoid of fibrin deposition lacked detectable VEGF immunoreactivity, whereas biopsies with fibrin deposition showed VEGF immunoreactivity in cardiocytes, interstitium, and some microvessels. By in situ hybridization, biopsies without microvascular fibrin deposition showed faint VEGF hybridization signals confined primarily to cardiocytes. In biopsies with fibrin deposition, strong VEGF hybridization signals were detected in cardiocytes, arteriolar smooth muscle cells were occasionally labeled, and endothelial cells were rarely labeled. By quantitative RT-PCR, biopsies with fibrin deposition (n=5) relatively expressed approximately three-fold more VEGF mRNA than biopsies without fibrin deposition (n=5 P=0.02). Serum VEGF titers also were greater (P=0.01) in recipients with fibrin deposition (372.9+/-66.7 pg/ml n=18) compared to recipients without fibrin deposition (172.1+/-25.0 pg/ml n=16). Collectively, these results support the hypothesis that increased myocyte-derived VEGF production following microvascular fibrin deposition in transplanted human hearts may act in a paracrine manner to promote activational and phenotypic changes in capillaries that provide a survival advantage for the allografts.

A study of the relationship between placenta growth factor and gestational age, parturition, rupture of membranes, and intrauterine infection.

OBJECTIVE: Placenta growth factor is a potent angiogenic factor produced by the human placenta that has been implicated in the pathogenesis of preeclampsia and intrauterine growth restriction. Placenta growth factor belongs to the vascular endothelial growth factor family and is capable of inducing proliferation, migration, and activation of endothelial cells. The objective of this study was to determine the relationship between amniotic fluid concentration of placenta growth factor and gestational age, parturition (term and preterm), spontaneous rupture of the membranes, and intra-amniotic infection. STUDY DESIGN: Amniotic fluid samples obtained from 273 pregnant patients were assayed in the following clinical groups: midtrimester pregnancy, preterm labor who delivered at term, preterm labor without microbial invasion of the amniotic cavity who delivered preterm, preterm labor with microbial invasion of the amniotic cavity, term not in labor, term in labor, term with microbial invasion of the amniotic cavity, preterm premature rupture of membranes with and without microbial invasion of the amniotic cavity, and term with premature rupture of membranes without microbial invasion of the amniotic cavity. The placenta growth factor concentrations were determined by an immunoassay that is both sensitive and specific. RESULTS: Placenta growth factor was detectable in 96.3% (263/273) of samples. Amniotic fluid placenta growth factor concentration decreased with advancing gestational age (r = -0.42; P <.001). Amniotic fluid placenta growth factor concentrations were significantly higher in women in midtrimester pregnancy than in those at term not in labor (midtrimester pregnancy: median, 43.1 pg/mL; range, 22.9-69.8 pg/mL; vs term not in labor: median, 28.7 pg/mL; range, 16.1-82.7 pg/mL; P <.01). Neither term nor preterm parturition was associated with a change in amniotic fluid placenta growth factor concentrations. Term premature rupture of membranes was associated with a significant decrease in amniotic fluid placenta growth factor concentration (term premature rupture of membranes: median, 16.5 pg/mL; range <5.2-195.1 pg/mL; vs term intact membranes: median, 28.7 pg/mL; range, 16.1-822.7 pg/mL; P <.005). Preterm premature rupture of membranes was not associated with changes in amniotic fluid placenta growth factor concentrations. Intra-amniotic infection in preterm labor, term labor with intact membranes, and preterm premature rupture of membranes were not associated with changes in amniotic fluid placenta growth factor concentrations. CONCLUSION: Placenta growth factor is a physiologic constituent of amniotic fluid. Amniotic fluid concentrations of placenta growth factor decrease with advancing gestational age. Neither parturition nor infection affects amniotic fluid placenta growth factor concentrations.

Signal transduction and biological function of placenta growth factor in primary human trophoblast.

Placenta growth factor (PlGF), a member of the vascular endothelial growth factor family of angiogenic factors, is prominently expressed by trophoblast. In addition to its role as a paracrine angiogenic factor within the placenta and endometrium, presence of its receptor, Flt-1, on trophoblast suggests that PlGF also may have an autocrine role(s) in regulating trophoblast function. To elucidate its role in trophoblast, we examined the signal transduction and functional responses of primary human trophoblast to PlGF. Exogenous PlGF induced specific activation of the stress-activated protein kinase (SAPK) pathways, c-Jun-N terminal kinase (JNK) and p38 kinase, in primary term trophoblast with little to no induction of the extracellular signal regulated kinase (ERK-1 and -2) pathways. In contrast, PlGF induced significant ERK-1 and -2 activity in human umbilical vein endothelial cells but did not induce JNK or p38 activity. PlGF-induced activation of the SAPK signaling pathways protected trophoblast from growth factor withdrawal-induced apoptosis, but it did not protect trophoblast from apoptosis induced by the pro-inflammatory cytokines, interferon gamma and tumor necrosis factor alpha. These results provide the first direct evidence of a biochemical and functional role for PlGF/Flt-1 in normal trophoblast and suggest that aberrant PlGF expression during pregnancy may impact upon trophoblast function as well as vascularity within the placental bed.

Placenta growth factor: potential role in pregnancy.

PROBLEM: In spite of the known requirement for adequate vascularity during placentation, little is known regarding the regulation of angiogenic growth factor production by trophoblast. Placenta growth factor (PIGF) is a recently discovered angiogenic growth factor whose expression is relatively limited to trophoblast. METHOD OF STUDY: Current literature of PIGF was reviewed, with emphasis on its expression, regulation, role in angiogenesis, and potential function(s) at the maternal-fetal interface. RESULTS: PIGF is abundantly expressed by trophoblast, which implies that it could act in a paracrine manner to modulate vascular development, stability, and/or function within the decidua and placental villi. In addition, expression of the PIGF receptor, fms-like tyrosine kinase (flt-1) receptor, on trophoblast raises the potential for an autocrine role of PIGF in regulating trophoblast growth and/or function. CONCLUSIONS: The potential for PIGF to influence both vascular endothelial cells and trophoblast suggests that aberrant trophoblast production of PIGF could compromise cellular function during gestation and contribute to the vascular and placental pathologies noted in many obstetric complications.

Preeclampsia is associated with reduced serum levels of placenta growth factor.

OBJECTIVES: Adequate vascular development of the placental bed is essential for normal pregnancy. We assessed serum levels of placenta growth factor, an angiogenic factor, throughout normal pregnancy and determined its association with preeclampsia. STUDY DESIGN: Serum samples were collected from (1) 308 healthy pregnant women throughout normal gestation, (2) at delivery from 30 each gestational age-matched patients with normal pregnancy and preeclampsia, and (3) maternal and cord blood samples from normal deliveries with and without labor (n = 37 each). Placenta growth factor levels were determined with an antigen-capture enzyme-linked immunosorbent assay. RESULTS: Maternal placenta growth factor levels during normal pregnancy increased from the first trimester to the late second trimester; they subsequently declined from 30 weeks' gestation to delivery. Significantly less maternal placenta growth factor (P <.0001) was found in pregnancies complicated by preeclampsia, and labor significantly lowered placenta growth factor levels in both maternal (P =.0189) and cord serum samples (P <.0001). CONCLUSION: Decreased levels of placenta growth factor during preeclampsia could influence endothelial cell and trophoblast function, thereby contributing to the pathogenesis of the disease.

Uteroplacental vascular involvement in recurrent spontaneous abortion.

Spontaneous abortion is common in human pregnancy. Recent advances in pregnancy immunology and vascular biology are reviewed with emphasis upon the events associated with recurrent fetal losses. Certain treatment options used to alleviate or prevent some miscarriages are presented and discussed.

Vascular endothelial growth factor, placenta growth factor and their receptors in isolated human trophoblast.

The expression of the angiogenic growth factors, vascular endothelial cell growth factor (VEGF) and placenta growth factor (PIGF) was demonstrated in isolated human term cytotrophoblast and in vitro differentiated syncytiotrophoblast. RNase protection assays demonstrated VEGF expression in both cytotrophoblast and syncytiotrophoblast while prominent PIGF expression was detected in both types of trophoblast by Northern blot analyses. VEGF expression increased approximately eightfold in trophoblast cultured under hypoxic conditions (1 per cent O2) yet PIGF expression decreased 73 +/- 5.5 per cent in the same trophoblast. These results suggest distinct regulatory mechanisms govern expression of VEGF and PIGF in trophoblast. Characterization of the VEGF/PIGF receptors, KDR and flt-1, revealed the presence of flt-1 mRNA in isolated cytotrophoblast and in vitro differentiated syncytiotrophoblast. KDR was not detected in the isolated trophoblast. Exogenous rhVEGF induced c-Jun N-terminal kinase (JNK) activity in the normal trophoblast indicating that the flt-1 receptors on trophoblast are functional. Trophoblast-derived VEGF/PIGF could act in a paracrine fashion to promote uterine angiogenesis and vascular permeability within the placental bed. In addition, presence of function flt-1 on normal trophoblast suggests that VEGF/PIGF functions in an autocrine manner to perform an as yet undefined role in trophoblast invasion, differentiation, and/or metabolic activity during placentation.

Is binding to nicotinic acetylcholine and dopamine receptors related to working memory in rats?

Nicotinic acetylcholine (ACh) and dopamine (DA) receptor activation has been found to be important for working memory. The regional distribution of these receptors in the brain has been well characterized. However, the relationship of the region-specific nicotinic ACh and DA binding density to memory performance has not been well assessed. In the current studies the relationship of receptor binding and memory function was examined. Receptor binding and memory performance were assessed in rats in three types of conditions: 1) chronic nicotine and mecamylamine vs. vehicle infusion; 2) lesions of the fimbria-fornix or medial basalocortical projection vs. sham lesions; and 3) 2-year-old aged rats vs. 3-month-old young adult rats. Nicotinic ACh receptors were labeled by [3H]N-methyl-carbamylcholine ([3H]MCC), D1 receptors by [3H]SCH 23390, and D2 receptors by [125I]iodosulpiride. Working memory was assessed using the radial-arm maze and T-maze delayed spatial alternation tasks. Chronic nicotine infusion substantially increased nicotinic receptor binding in a variety of brain areas and significantly improved working memory performance in the radial-arm maze. However, nicotinic receptor binding did not correlate well with memory performance. The nicotinic antagonist mecamylamine did not block nicotine-induced increased nicotinic binding, but it did block nicotine-induced memory improvement. Aged rats relative to young adults showed both a decrease in nicotinic binding and impaired memory performance. However, chronic effects of nicotine on nicotinic receptor binding and memory performance did not correlate in the aged rats. Nicotine also increased nicotinic receptor binding in the aged rats in brain areas except for the VTA, but did not improve memory performance. Lesions of the medial basalocortical projection or the fimbria-fornix did not cause significant changes in nicotinic binding in their target fields, but they did cause significant deficits in memory performance. Finally, there were no significant correlations of nicotinic binding in any brain region and memory performance. DA receptor binding was not altered by chronic nicotine or mecamylamine infusion, fimbria-fornix lesions, medial basalocortical lesions, or in aged rats. However, DA receptor binding did correlate with memory performance. There was a positive correlation of T-maze accuracy and D1 receptor binding in the frontal cortex and a negative correlation of T-maze accuracy and D1 receptor binding in the VTA and dentate gyrus. In contrast, a positive correlation was seen between radial-arm maze accuracy and D1 receptor binding in the VTA. Radial-arm maze accuracy was positively correlated with D2 receptor binding in the striatum and dentate gyrus. There are significant relationships between the extent of DA receptor binding and working memory, but relationship between nicotinic ACh receptor binding density and memory is weak.

Angiogenesis and the expression of vascular endothelial growth factor in endometrium and placenta.

PROBLEM: The demand for increased angiogenesis and microvascular permeability during cyclical changes in the endometrium and during placentation raises the possibility that aberrations in these events could lead to suboptimal reproductive performance. However, relatively little is presently known regarding the regulation of vascular growth and permeability in these tissues. METHOD OF STUDY: This review of current literature focuses on the expression, regulation, and potential physiological effects of vascular endothelial growth factor (VEGF) within endometrial and placental tissue. RESULTS: Spatial and temporal expression of VEGF as well as its restricted specificity, essential role in vasculogenesis/angiogenesis, and ability to induce vascular permeability makes VEGF an attractive regulator of vascular growth and permeability in the endometrium and placenta. CONCLUSION: A better understanding of the production, regulation, and physiological responses of the vasculature to angiogenic growth factors may lead to new therapeutic strategies for reproductive disturbances secondary to vascular insufficiencies within the female reproductive tract.

Overexpression of RANTES using a recombinant adenovirus vector induces the tissue-directed recruitment of monocytes to the lung.

RANTES (regulated on activation, normal T cell expressed and secreted) is a member of the C-C superfamily of chemokines and is reported to function as a potent chemoattractant for monocytes, eosinophils, and a subpopulation of CD4+ T cells. Using a recombinant human type 5 adenovirus containing the murine RANTES cDNA (Ad5E3 mRANTES), which is capable of expressing biologically active cytokine upon infection, we initiated a study to characterize the biologic functions of RANTES cytokine in vivo. Intratracheal administration of Ad5E3 mRANTES targeted transient RANTES expression to the bronchial epithelium of the lung in Sprague-Dawley rats. Bronchoalveolar lavage fluids (BAL) collected at 24 h had increased chemotactic activity vs controls as measured in a murine CD4+ T cell Boyden chamber microchemotaxis assay. There was a dramatic increase in the number of cells (macrophage, monocytes, and neutrophils) recovered from BAL samples taken from Ad5E3 mRANTES-treated animals at 24 h, with a >50-fold increase in monocytes, indicating a proinflammatory effect for this cytokine in vivo. This effect on monocytes was transient, decreasing by 7 days, with evidence of increased eosinophils and lymphocytes at this time. Histologic examination of lung sections at 24 h revealed greatly increased numbers of mononuclear cells, primarily monocytes, within the lungs of Ad5E3 mRANTES-treated animals, with increased extravasation of monocytes around blood vessels, indicating an ongoing process of peripheral blood monocyte recruitment. This study provides further evidence for RANTES to be a monocyte chemoattractant in vivo.

Vascular endothelial growth factor expression in cycling human endometrium.

OBJECTIVE: To determine the spatial distribution of vascular endothelial growth factor protein in human endometrium and to assess temporal fluctuations in vascular endothelial growth factor gene expression and variant isoform production by stromal and epithelial cells during the menstrual cycle. DESIGN: Prospective study design. PATIENTS: Early proliferative endometrial biopsies were obtained from women undergoing gynecologic surgery for benign conditions; secretory stage biopsies were obtained from patients undergoing routine infertility investigations without evidence of luteal insufficiency. MAIN OUTCOME MEASURE: Immunohistochemical detection of vascular endothelial growth factor protein in endometrial biopsies, analyses of vascular endothelial growth factor RNA expression, and isoform production in intact endometrium and isolated endometrial stromal and epithelial cells. RESULTS: Strong vascular endothelial growth factor immunoreactivity was detected in the glandular epithelial cells of the secretory endometrium with no discernible immunoreactivity in stroma cells. The proliferative endometrium demonstrated prominent glandular immunoreactivity and faint, inconsistent stromal cell immunoreactivity. Preincubation of the antibody with excess cognate peptide abolished all immunoreactivity. A threefold to sixfold increase in vascular endothelial growth factor messenger RNA expression occurs in secretory versus proliferative endometrial samples. Endometrial stromal and epithelial cell isolates from both phases of the menstrual cycle express VEGF121, VEGF165, and VEGF189 isoforms, however, vascular endothelial growth factor variant 206 was not detected. CONCLUSIONS: Expression of vascular endothelial growth factor in the endometrium throughout the menstrual cycle suggests that vascular endothelial growth factor may promote the vascular growth, maintenance, and hyperpermeability required for adequate receptivity in the cycling human endometrium.

Modulation of the anchorage-independent phenotype of human lung fibroblasts obtained from fibrotic tissue following culture with retinoid and corticosteroid.

Fibroblast heterogeneity has been documented in fibrotic tissue from lung and skin. Differences have been demonstrated in proliferative rates in fibroblasts derived from fibrotic lung tissue as compared to normal. Fibroblast lines derived from adult fibrotic lung tissue and neonatal normal lung tissue exhibit colony growth in soft agarose culture, whereas fibroblast cell lines from normal adult lung tissue do not. The characteristic of anchorage-independent growth is consistent with the aggressive nature of the disease and with developmental lung growth. In this study, fibrotic lung fibroblasts were exposed to growth and differentiating factors to determine whether the anchorage-independent phenotype can be modulated. The results indicate that treatment of fibrotic lung fibroblasts with retinoic acid, known to modify matrix gene expression and induce differentiation, inhibits the cells ability to form colonies under soft agarose growth. Treatment with all-trans-retinoic acid yielded the greatest effect inhibiting both IPF and neonatal lung fibroblast anchorage-independent growth approximately 90% at 10(-6) M. Treatment of IPF fibroblasts with all-trans-retinoic acid also inhibited corticosteroid-induced colony growth. Modulation of the "fibrotic" fibroblast phenotype through retinoid therapy may prove beneficial as a potential therapeutic strategy.

Acute and chronic nicotine effects on working memory in aged rats.

Acute and chronic nicotine administration has been repeatedly been found in our laboratory to improve working memory performance of normal adult rats in the radial-arm maze. The current study was conducted to determine if acute or chronic nicotine administration would improve working memory performance in aged rats. Sixteen young adult (3-7 months) and 32 aged (24-28 months) male Sprague-Dawley rats were trained on an eight-arm radial maze. A significant age-related choice deficit was seen during the 21 sessions of training. After training, half of the rats in each age group were implanted with nicotine-containing osmotic minipumps and the other half implanted with vehicle-containing pumps. Consistent with previous work, the young adult rats given chronic nicotine (approximately 5 mg/kg per day as measured as nicotine base) showed a significant improvement in working memory performance. In contrast, the aged rats did not show a significant effect of this dose of chronic nicotine. After a 2 week withdrawal period the remaining rats underwent a series of acute drug challenges with nicotinic and muscarinic agonists and antagonists as well as the dopaminergic antagonist haloperidol. Mecamylamine and haloperidol impaired the memory performance of the young adult rats, whereas the aged rats showed no effect. In contrast, scopolamine impaired performance of both young adult and aged rats in a similar manner. Both pilocarpine and nicotine improved the memory performance of the aged rats, but did not improve the young adult rats, possibly due to a ceiling effect on performance. During the cholinergic agonist drug phase, the aged rats which had previously been given chronic nicotine infusions showed better performance than those which had not. The resistance of the aged rats to chronic nicotine-induced working memory improvements and acute mecamylamine-induced working memory deficits may have resulted from the decline in nicotinic receptors seen with aging. Chronic co-administration of the nicotinic antagonist mecamylamine in a previous study was found to abolish the chronic nicotine-induced working memory improvement. The aged rats were resistant to haloperidol-induced deficits which may have resulted from the decrease in dopaminergic receptors seen with aging. Interestingly, acute cholinergic agonists including nicotine did improve working memory performance in the aged rats and previous chronic nicotine infusion was beneficial during the period of acute cholinergic agonist challenge. This suggests that nicotinic treatment may be of use for treating age associated memory impairments but that special dosing regimens may be required.

IL-1 beta, TNF-alpha, and IL-2 in peritoneal fluid and macrophage-conditioned media of women with endometriosis.

PROBLEM: The presence of various cytokines in human peritoneal fluid has been incompletely evaluated. Changes in cytokine levels may be related to the development of endometriosis, infertility, and activation of peritoneal macrophages. This study assesses levels of IL-1 beta, IL-2 and TNF- alpha in peritoneal fluid and macrophage conditioned media of women with endometriosis. METHOD: Peritoneal fluid was collected from 51 women at the time of diagnostic or operative laparoscopy for benign gynecologic disease. Peritoneal macrophages were isolated, cultured for 24 h, and the culture media collected. IL-1 beta, IL-2, and TNF- alpha levels were determined by commercial ELISA kits. RESULTS: The mean concentration of IL-1 beta and TNF- alpha was significantly higher in macrophage conditioned media of patients with endometriosis (P < 0.02). However, there were no significant changes in peritoneal fluid cytokine levels. Peritoneal macrophage concentrations were also higher in patients with endometriosis. CONCLUSION: This study supports the concept that endometriosis is associated with activation of peritoneal macrophages, and a higher concentration of these cells. This activation is reflected by the increased levels of cytokines found in macrophage conditioned media. The absence of significant changes in peritoneal fluid cytokine levels would seen to indicate that the above derangements are not responsible for the development or progression of endometriosis.

Vascular endothelial growth factor expression in transplanted human hearts.

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen thought to play an important role in coronary collateral vessel formation. We used immunocytochemistry to determine VEGF expression in biopsies (n = 283) of transplanted human hearts (n = 109) with and without microvascular fibrin. Measures of vascular fibrin, alpha 2 plasmin-inhibitor (a2Pl), macrophages, neutrophils, and serum cardiac troponin T titers were used to evaluate myocardial damage. Antibody to T lymphocytes was used to evaluate cellular rejection, and HLA-DR, ICAM-1, and PAL-E antibodies were used to assess endothelial cell activation and phenotypic changes in the microcirculation. No VEGF immunoreactivity was detected in control donor hearts without fibrin, but the proportion of biopsies demonstrating VEGF immunoreactivity increased significantly in allografts with increasing fibrin and a2PI reactivity (P = 0.0001). VEGF immunoreactivity was confined to areas of fibrin deposition and was associated with infiltrates of macrophages and neutrophils (P < 0.0001), but not with T cells (P = 0.10). Biopsies with fibrin/VEGF reactivity were associated with increased capillary endothelial cell HLA-DR, ICAM-1, and PAL-E reactivity. In a subset of patients, serum cardiac troponin-T values were greater in patients with VEGF-positive (n = 21) than VEGF-negative (n = 19) biopsies (P = 0.05). Nested RT-PCR demonstrated that biopsies with and without fibrin/VEGF immunoreactivities expressed VEGF121, VEGF165, and VEGF189 variants, with VEGF165 being the dominate variant. These results indicate that endogenous VEGF is expressed locally following vascular thrombosis and myocardial cell damage, and that VEGF expression may be related to endothelial cell activation and phenotypic changes found in the microcirculation of cardiac allografts.