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Tay-Sachs (TS) disease is a neurodegenerative disease resulting from mutations in the gene encoding the α-subunit (HEXA) of lysosomal β-hexosaminidase A (HexA). We report that (1) recombinant HEXA alone increased HexA activity and decreased GM2 content in human TS glial cells and peripheral mononuclear blood cells; 2) a recombinant chimeric protein composed of HEXA linked to two blood-brain barrier (BBB) entry elements, a transferrin receptor binding sequence and granulocyte-colony stimulating factor, associates with HEXB in vitro ; reaches human cultured TS cells lysosomes and mouse brain cells, especially neurons, in vivo ; lowers GM2 in cultured human TS cells; lowers whole brain GM2 concentration by approximately 40% within 6 weeks, when injected intravenously (IV) to adult TS-mutant mice mimicking the slow course of late-onset TS; and increases forelimbs grip strength. Hence, a chimeric protein equipped with dual BBB entry elements can transport a large protein such as HEXA to the brain, decrease the accumulation of GM2, and improve muscle strength, thereby providing potential treatment for late-onset TS., Competing Interests: N.Stern, E.O., O.B., and Y.A. are inventors in patent WO 2022/180628. N.Stern, E.O., and R.N. are inventors in patent US20100183577A1., (© 2024 The Authors.)

Objective: The early embryo implantation is characterized by enhanced uterine vascular permeability at the site of blastocyst attachment, followed by extracellular-matrix remodeling and angiogenesis. Two TG (transglutaminase) isoenzymes, TG2 (tissue TG) and FXIII (factor XIII), catalyze covalent cross-linking of the extracellular-matrix. However, their specific role during embryo implantation is not fully understood. Approach and Results: For mapping the distribution as well as the enzymatic activities of TG2 and FXIII towards blood-borne and resident extracellular-matrix substrates, we synthetized selective and specific low molecular weight substrate analogs for each of the isoenzymes. The implantation sites were challenged by genetically modifying the trophoblast cells in the outer layer of blastocysts, to either overexpress or deplete TG2 or FXIII, and the angiogenic response was studied by dynamic contrast-enhanced-magnetic resonance imaging. Dynamic contrast-enhanced-magnetic resonance imaging revealed a decrease in the permeability of decidual vasculature surrounding embryos in which FXIII were overexpressed in trophoblast cell. Reduction in decidual blood volume fraction was demonstrated when either FXIII or TG2 were overexpressed in embryonic trophoblast cell and was elevated when trophoblast cell was depleted of FXIII. These results were corroborated by histological analysis., Conclusions: In this study, we report on the isoenzyme-specific roles of TG2 and FXIII during the early days of mouse pregnancy and further reveal their involvement in decidual angiogenesis. Our results reveal an important magnetic resonance imaging-detectable function of embryo-derived TG2 and FXIII on regulating maternal angiogenesis during embryo implantation in mice.Visual Overview: An online visual overview is available for this article.

Background: Sorafenib improves progression-free survival in patients with progressive radioactive iodine-refractory differentiated thyroid carcinoma, but causes severe side effects. Estrogens may accelerate thyroid carcinoma cell growth. Our group recently reported that isoflavone derivative 7-(O)-carboxymethyl daidzein conjugated to N-t-boc-hexylenediamine (cD-tboc), a novel anti-estrogenic compound, retards the growth of both thyroid carcinoma cell lines and cultured human carcinoma cells. Vitamin D receptor (VDR) is expressed in malignant cells and responds to 1,25 dihydroxyvitamin D3 (1.25D) by decreased proliferative activity in vitro. The purpose of this study was to examine the effects of vitamin D metabolites (VDM) on the expression of estrogen receptors (ERs), VDR, and 1OHase mRNA, and to evaluate the inhibitory effect of low doses of sorafenib in combination with cDtboc and VDM on cell proliferation in cultured human papillary thyroid carcinoma (PTC)., Methods: In 19 cultured PTC specimens and 19 normal thyroid specimens, harvested during thyroidectomies from the same patients, expression levels of ERα, ERβ, VDR, and 1 alpha-hydroxylase (1OHase) mRNA (by quantitative real-time PCR) were determined at baseline and after treatment with VMD. Cell proliferation was determined by measurement of 3 [H] thymidine incorporation after treatment with sorafenib alone, sorafenib with added 1.25D or cD-tboc, and sorafenib with both 1.25D and cD-tboc added., Results: 1,25D increased mRNA expression of all tested genes in the malignant and normal thyroid cells, while the ERα mRNA of the normal cells was unaffected. 1.25D dose-dependently inhibited cell proliferation in the malignant cells. The inhibitory effect of sorafenib on cell proliferation in the malignant cells was amplified after the addition of cDtboc and 1.25D, such that the maximal inhibition was not only greater, but also had been attained at a 10-fold lower concentration of sorafenib (20 μg/ml). This inhibition was similar to that of the generally used concentration of sorafenib (200 μg/ml) alone., Conclusions: The demonstration that low concentrations of cDtboc and 1.25D markedly amplify the inhibitory effect of sorafenib on the growth of human PTC supports the use of a 10-fold lower concentration of sorafenib. The findings may promote a new combination treatment for progressive radioactive iodine-refractory PTC., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Hyaluronan is a biologically active polymer, which can be formulated into nanoparticles. In our study, we aimed to probe atherosclerosis-associated inflammation by using hyaluronan nanoparticles and to determine whether they can ameliorate atherosclerosis. Hyaluronan nanoparticles (HA-NPs) were prepared by reacting amine-functionalized oligomeric hyaluronan (HA) with cholanic ester and labeled with a fluorescent or radioactive label. HA-NPs were characterized in vitro by several advanced microscopy methods. The targeting properties and biodistribution of HA-NPs were studied in apoe -/- mice, which received either fluorescent or radiolabeled HA-NPs and were examined ex vivo by flow cytometry or nuclear techniques. Furthermore, three atherosclerotic rabbits received 89 Zr-HA-NPs and were imaged by PET/MRI. The therapeutic effects of HA-NPs were studied in apoe -/- mice, which received weekly doses of 50 mg/kg HA-NPs during a 12-week high-fat diet feeding period. Hydrated HA-NPs were ca. 90 nm in diameter and displayed very stable morphology under hydrolysis conditions. Flow cytometry revealed a 6- to 40-fold higher uptake of Cy7-HA-NPs by aortic macrophages compared to normal tissue macrophages. Interestingly, both local and systemic HA-NP-immune cell interactions significantly decreased over the disease progression. 89 Zr-HA-NPs-induced radioactivity in atherosclerotic aortas was 30% higher than in wild-type controls. PET imaging of rabbits revealed 6-fold higher standardized uptake values compared to the muscle. The plaques of HA-NP-treated mice contained 30% fewer macrophages compared to control and free HA-treated group. In conclusion, we show favorable targeting properties of HA-NPs, which can be exploited for PET imaging of atherosclerosis-associated inflammation. Furthermore, we demonstrate the anti-inflammatory effects of HA-NPs in atherosclerosis.

Background: Deep dysphasia is a relatively rare subcategory of aphasia, characterised by word repetition impairment and a profound auditory-verbal short-term memory (STM) limitation. Repetition of words is better than nonwords (lexicality effect) and better for high-image than low-image words (imageability effect). Another related language impairment profile is phonological dysphasia, which includes all of the characteristics of deep dysphasia except for the occurrence of semantic errors in single word repetition. The overlap in symptoms of deep and phonological dysphasia has led to the hypothesis that they share the same root cause, impaired maintenance of activated representation of words, but that they differ in severity of that impairment, with deep dysphasia being more severe., Aims: We report a single-subject multiple baseline, multiple probe treatment study of a person who presented with a pattern of repetition that was consistent with the continuum of deep-phonological dysphasia: imageability and lexicality effects in repetition of single and multiple words and semantic errors in repetition of multiple-word utterances. The aim of this treatment study was to improve access to and repetition of low-imageability words by embedding them in modifier-noun phrases that enhanced their imageability., Methods & Procedures: The treatment involved repetition of abstract noun pairs. We created modifier-abstract noun phrases that increased the semantic and syntactic cohesiveness of the words in the pair. For example, the phrases "long distance" and "social exclusion" were developed to improve repetition of the abstract pair "distance-exclusion". The goal of this manipulation was to increase the probability of accessing lexical and semantic representations of abstract words in repetition by enriching their semantic -syntactic context. We predicted that this increase in accessibility would be maintained when the words were repeated as pairs, but without the contextual phrase., Outcomes & Results: Treatment outcomes indicated that increasing the semantic and syntactic cohesiveness of low-imageability and low-frequency words later improved this participant's ability to repeat those words when presented in isolation., Conclusions: This treatment approach to improving access to abstract word pairs for repetition was successful for our participant with phonological dysphasia. The approach exemplifies the potential value in manipulating linguistic characteristics of stimuli in ways that improve access between phonological and lexical-semantic levels of representation. Additionally, this study demonstrates how principles of a cognitive model of word processing can be used to guide treatment of word processing impairments in aphasia., Competing Interests: Disclosure statement No potential conflict of interest was reported by the authors.

The net vascular effect of estrogens on the vasculature is still under debate. Here we tested the effects of estradiol- 17β (E2) as well as estrogen-receptor subtype specific and non-specific agonists and antagonists on the expression and eicosanoid production of lipoxygenase (LO) enzymes expressed in culture human umbilical vascular smooth muscle cells (VSMC), the platelet type 12LO and 15LO type 2. E2 increased 12 and 15LO mRNA expression by 2-3 folds and elicited an acute 50% increase 12 and 15 hydroxyeicosatetraenoic acid (HETE) production. Neither estrogen receptor ERα nor ERβ-specific agonists were able to reproduce the induction of LO expression, but E2-induced expression was effectively blocked by ER non-specific and receptor subtype specific antagonists. Because 12 and 15HETE can increase reactive oxygen species in other cell types, we tested the possibility that E2 could raise ROS through LO. Indeed, E2 as well as the LO products 12 and 15HETE increased reactive oxygen species (ROS) in VSMC. E2-dependent and HETE-induced ROS could be blocked by NAD (P) H-oxidase inhibitors and by the ER general antagonist ICI. E2-induced ROS was partially (∼50%) blocked by the LO inhibitor baicalein, but the LO blocker had no effect on 12 or 15HETE- induced ROS formation, thus suggesting that part of E2-dependent ROS generation resulted from E2-induced 12 and 15HETE. Collectively these findings unveil an unrecognized effect of E2 in human VSMC, to induce 12 and 15LO type 2 expression and activity and suggest that E2-dependent ROS formation in VSMC may be partially mediated by the induction of 12 and 15HETE., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

CGβ subunits comprise a unique carboxyl-terminal peptide (CTP) that has multiple O-linked glycans and extends serum half-life of the protein. It has evolved by incorporating a previously untranslated region of the LHβ gene into the reading frame. Although CTP-like sequences are encrypted in the LHβ genes of several mammals, the CGβ subunit developed only in primates and equids. To study this restriction in evolution, we examined whether the cryptic CTP decoded from the bovine LHβ gene (boCTP) possesses key characteristics of the human (h) CGβ-CTP. The boCTP does not impede several crucial aspects of hormone biosynthesis, but compared to the hCGβ-CTP, the stretch lacks O-glycans and determinants for circulatory survival. O-glycan deficiency and the associated incapacity to extend serum half-life is a major drawback of the boCTP. This may explain why LH did not evolve into CG in ruminants and consequently alternative mechanisms evolved to delay luteolysis early in gestation., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

We studied the effects of chronic angiotensin 1-7 (Ang 1-7) treatment in an experimental model of the metabolic syndrome, i.e., rats given high-fructose/low-magnesium diet (HFrD). Rats were fed on HFrD for 24 weeks with and without Ang 1-7 (576 µg/kg/day, s.c., Alzet pumps). After 6 months, Ang 1-7-treated animals had lower body weight (-9.5%), total fat mass (detected by magnetic resonance imaging), and serum triglycerides (-51%), improved glucose tolerance, and better insulin sensitivity. Similar metabolic effects were also evident, albeit in the absence of weight loss, in rats first exposed to HFrD for 5 months and then subjected to short-term (4 weeks) treatment with Ang 1-7. Six months of Ang 1-7 treatment were associated with lower plasma renin activity (-40%) and serum aldosterone (-48%), less hepatosteatatitis, and a reduction in epididymal adipocyte volume. The marked attenuation of macrophage infiltration in white adipose tissue (WAT) was associated with reduced levels of the pP65 protein in the epididymal fat tissue, suggesting less activation of the nuclear factor-κB (NFκB) pathway in Ang 1-7-treated rats. WAT from Ang 1-7-treated rats showed reduced NADPH-stimulated superoxide production. In single muscle fibers (myofibers) harvested and grown ex vivo for 10 days, myofibers from HFrD rats gave rise to 20% less myogenic cells than the Ang 1-7-treated rats. Fully developed adipocytes were present in most HFrD myofiber cultures but entirely absent in cultures from Ang 1-7-treated rats. In summary, Ang 1-7 had an ameliorating effect on insulin resistance, hypertriglyceridemia, fatty liver, obesity, adipositis, and myogenic and adipogenic differentiation in muscle tissue in the HFrD rats.

Currently available treatments for patients with medullary thyroid carcinoma (MTC) with residual or recurrent disease after primary surgery have low efficacy rates. In view of the possible role of estrogen in the development of thyroid neoplasia, we explored whether proliferation of the human MTC TT cell line, might be curbed by carboxy-daidzein-tBoc (cD-tBoc), a novel isoflavone derivative. Estrogen receptor (ER) α mRNA expression in TT cells was more abundant than ERβ, with a ratio of 48:1. Estradiol-17β (E2) increased DNA synthesis in a dose dependent manner. [(3)H]-thymidine incorporation was also stimulated by the ERβ agonist DPN and the ERα agonist PPT. cD-tBoc inhibited TT cell growth as assessed by thymidine incorporation, XTT assay, and microscopic analysis of culture wells. Creatine kinase specific activity, a marker of the modulatory effects of estrogen on cell energy metabolism, was likewise inhibited. The inhibitory effect of cD-tBoc on [(3)H]-thymidine incorporation could be blocked by the ERβ antagonist PTHPP but not by the ERα antagonist MPP, suggesting that the antiproliferative effect of cD-tBoc on these cells is mediated through ERβ. Furthermore, cD-tBoc potently increased apoptosis and cell necrosis. Co-incubation with the antiapoptotic agent Z-VAD-FMK reversed the growth inhibitory effect elicited by cD-tBoc. These results support the hypothesis that estrogens are involved in the proliferation of MTC. The potent anti-proliferative effects mediated by isoflavone derivatives in the human MTC cell line TT suggest and that this property may be utilized to design effective anti-neoplastic agents., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Estrogens may enhance thyroid cancer cell growth. We have recently reported that a novel isoflavone-derived anti-estrogenic compound developed in our laboratory, the N-t-boc-hexylenediamine derivative of 7-(O)-carboxymethyl daidzein (cD-tboc), can induce apoptosis and retard growth in human thyroid carcinoma cell lines through inhibitory interaction on estrogen receptor β. Here we tested the hypothesis that cD-tboc can likewise retard cell growth in cultured human thyroid papillary carcinoma cells, normal thyroid cells, and goiter cells removed during thyroidectomy., Methods: In vitro experiments in cultured human thyroid normal, goiter, and papillary thyroid carcinoma (PTC) cells were performed. Estrogen receptors α and β (ERα and ERβ), DNA synthesis and creatine kinase (a marker of estrogenic genomic response), and the effects of cD-tboc on DNA synthesis in cultured human PTC cells were assessed., Results: First, all cell types thus harvested and grown in culture expressed both ERα and ERβ, with a variably higher abundance of ERβ over ERα seen in the goiter and PTC cells, but not in the normal thyroid cells. Second, DNA synthesis and creatine kinase were increased in response to estradiol-17β (E2), the ERα agonist propyl-pyrazole-trisphenol as well as the ERβ agonist diarylpropionitrile. Third, cD-tboc dose-dependently inhibited DNA synthesis in cultured human PTC cells (-65%) and to a lesser extent in goiter cells (∼-30%)., Conclusion: This study provides the first evidence that cD-tboc can act to inhibit growth in primary cultures of human PTC cells and goiter cells removed during thyroidectomy. Whether this can be utilized for the treatment of human thyroid cancer and/or goiter remains to be explored.

BACKGROUND: Language performance in aphasia can vary depending on several variables such as stimulus characteristics and task demands. This study focuses on the degree of verbal working memory (WM) load inherent in the language task and how this variable affects language performance by individuals with aphasia. AIMS: The first aim was to identify the effects of increased verbal WM load on the performance of judgments of semantic similarity (synonymy) and phonological similarity (rhyming). The second aim was to determine if any of the following abilities could modulate the verbal WM load effect: semantic or phonological access, semantic or phonological short-term memory (STM) and any of the following executive processing abilities: inhibition, verbal WM updating, and set shifting. METHOD AND PROCEDURES: Thirty-one individuals with aphasia and 11 controls participated in this study. They were administered a synonymy judgment task and a rhyming judgment task under high and low verbal WM load conditions that were compared to each other. In a second set of analyses, multiple regression was used to identify which factors (as noted above) modulated the verbal WM load effect. OUTCOME AND RESULTS: For participants with aphasia, increased verbal WM load significantly reduced accuracy of performance on synonymy and rhyming judgments. Better performance in the low verbal WM load conditions was evident even after correcting for chance. The synonymy task included concrete and abstract word triplets. When these were examined separately, the verbal WM load effect was significant for the abstract words, but not the concrete words. The same pattern was observed in the performance of the control participants. Additionally, the second set of analyses revealed that semantic STM and one executive function, inhibition ability, emerged as the strongest predictors of the verbal WM load effect in these judgment tasks for individuals with aphasia. CONCLUSIONS: The results of this study have important implications for diagnosis and treatment of aphasia. As the roles of verbal STM capacity, executive functions and verbal WM load in language processing are better understood, measurements of these variables can be incorporated into our diagnostic protocols. Moreover, if cognitive abilities such as STM and executive functions support language processing and their impairment adversely affects language function, treating them directly in the context of language tasks should translate into improved language function.

Human ovarian cancer cells specifically bind the isoflavone daidzein. A chemical conjugate between daidzein and the garlic enzyme alliinase was prepared. The conjugate specifically bound to ovarian cancer cells and upon addition of the prodrug alliin, it effectively produced cytotoxic allicin molecules which killed the cancer cells. In vivo targeting and antitumor effect was confirmed by NIR and bioluminescence imaging using daidzein-alliinase-CyTE-777 conjugates and luciferase-expressing ovarian cancer cells. Co-localization of the fluorescent conjugate with bioluminescence was observed for intraperitoneal tumors while nonconjugated alliinase did not accumulate. Biodistribution studies with Europium-labeled conjugate revealed a five fold higher uptake in tumors as compared to other tissues. Treatment of tumor bearing mice with daidzein-alliinase and alliin effectively attenuated tumor progression during the first 12 days while a 5-fold increase in bioluminescence was detected in placebo-treated animals. Autopsy revealed only small individual foci of luminescence at the site of tumor cells inoculation. Histological examination of organs and tissues did not reveal any additional foci of carcinoma or signs of toxicity. These results suggest that the targeted alliinase conjugates in the presence of alliin, generated therapeutically effective levels of allicin which were capable of suppressing tumor progression of intraperitoneal ovarian cancer in an animal model.

BACKGROUND: Verbal short-term memory (STM) impairments are invariably present in aphasia. Word processing involves a minimal form of verbal STM, i.e., the time course over which semantic and phonological representations are activated and maintained until they are comprehended, produced, or repeated. Thus it is reasonable that impairments of word processing and verbal STM may co-occur. The co-occurrence of language and STM impairments in aphasia has motivated an active area of research that has revealed much about the relationship of these two systems and the effect of their impairment on language function and verbal learning (Freedman & Martin, 2001; Martin & Saffran, 1999; Trojano & Grossi, 1995). In keeping with this view a number of researchers have developed treatment protocols to improve verbal STM in order to improve language function (e.g., Koenig-Bruhin & Studer-Eichenberger, 2007). This account of aphasia predicts that treatment of a fundamental ability, such as STM, which supports language function, should lead to improvements that generalise to content and tasks beyond those implemented in treatment. AIMS: We investigated the efficacy of a treatment for language impairment that targets two language support processes: verbal short-term memory (STM) and executive processing, in the context of a language task (repetition). We hypothesised that treatment of these abilities would improve repetition abilities and performance on other language tasks that require STM. METHOD: A single-participant, multiple-baseline, multiple-probe design across behaviours was used with a participant with conduction aphasia. The treatment involved repetition of words and nonwords under three "interval" conditions, which varied the time between hearing and repeating the stimulus. Measures of treatment effects included acquisition, maintenance, and follow-up data, effect sizes, and pre- and post-treatment performance on a test battery that varies the STM and executive function demands of language tasks. OUTCOMES #ENTITYSTARTX00026; RESULTS: Improvement of repetition was mostly specific to treated stimuli. Post-treatment measures of language ability indicated improvements in single and multiple word processing tasks, verbal working memory tasks, and verbal span. CONCLUSIONS: Treatment of STM and executive processes in the context of a word repetition task resulted in improvements in other non-treated language tasks. The approach used in this study can be incorporated into other language-processing tasks typically used in treatment of language disorders (e.g., sentence processing).

Background: Sentence production impairment in aphasia has been attributed to several possible sources that are not mutually exclusive. Linguistic accounts often attribute the difficulty to the complexity of a verb's syntactic and/or semantic argument structure. Cognitive processing accounts emphasise the reduced processing capacity observed in agrammatic aphasia, which in turn has been attributed to reduced semantic short-term memory (STM) or slowed processing., Aims: In this study we used verb particles and prepositions to investigate effects of differences in syntactic and semantic argument structure on sentence repetition in aphasia. We predicted that verb particles and sentences containing verb-particle constructions would be easier to repeat than prepositions and prepositional transitive sentences, as the former have a less-complex semantic and syntactic argument structure than the latter. Also, semantic and phonological spans were assessed to determine if a reduction in either capacity correlates with repetition ability., Methods & Procedures: Participants were eight individuals with chronic aphasia. The experimental task was repetition of transitive sentences balanced for length and lexical content containing either verb particles or prepositional object structures. Accuracy of sentence repetition and repetition of verb particles and prepositions within sentences was examined. We calculated the effect of structural complexity on the sentence repetition task as the difference between proportion correct of verb-particle constructions and prepositional transitives. Semantic and phonological STM spans and word spans were also assessed and correlated with this measure of the structural complexity effect on sentence repetition., Outcomes & Results: Verb-particle sentences were repeated correctly significantly more often than prepositional transitive sentences, and within those sentences verbal particles were repeated correctly significantly more often than prepositions. The effect was strongly associated with fluency scores: it was present in participants with low fluency scores, but not in those with high fluency scores. The phonological, but not the semantic, STM probe span measure correlated with both the difference in accurate repetition of verb-particle and prepositional transitive sentences and the particles and prepositions within those sentences., Conclusions: Results indicate that differences in argument structure of particle and preposition constructions influence sentence repetition in agrammatic aphasia. The finding that lower fluency scores are associated with poorer performance on more complex structures suggests that this effect is associated with agrammatism. The impact of these structural distinctions between particles and prepositions should be taken into account during development of treatment stimuli for those with agrammatism.

One of the major difficulties in the treatment of epithelial ovarian cancer (EOC) is the high rate of recurrent disease. This is thought to be due to the survival of a population of chemo-resistant cells within the tumor, the ovarian cancer stem cells (OCSCs), that are able to regenerate the tumor following chemotherapy. Therefore, the identification of a compund that can target the OCSCs is one of the main steps in improving overall survival of ovarian cancer patients. The objective of this study was to determine the effect of N-t-boc-Daidzein, a novel daidzain derivative, on OCSCs. The efficacy of this compound was evaluated in OCSC and mature ovarian cancer cell (mOCC) lines isolated from malignant ovarian cancer asicites. Cells were treated with increasing concentrations of N-t-boc-Daidzein (0.003-10 microM) and cell growth was monitored by "real time in vitro micro-imaging" using the IncuCyte system. Cell viability was measured using the CellTiter 96 Assay. Apoptosis was determined by Caspase-Glo 3/7, 8 and 9 assays. The components of the apoptotic cascade were characterized by western blot analysis. N-t-boc-Daidzein was able to significantly inhibit cell growth and decrease cell viability of OCSC as well as mOCC cells in a dose and time dependent maner. This effect was due to the induction of apoptosis, which is characterized by caspase activation, XIAP and AKT degradation, and mitochondrial depolarization. This study describes a novel compound that can target the OCSCs. These findings may provide vital aide in improving overall survival in patients with EOC.

Ovarian cancer is the most lethal gynecologic malignancy, often diagnosed at advanced stage leading to poor prognosis. In the study reported here, magnetic resonance imaging and near-infrared reflectance imaging were applied for in vivo analysis of two competing endocytic pathways affecting retention of bifunctional daidzein-bovine serum albumin (BSA)-based contrast media by human epithelial ovarian carcinoma cells. Suppression of caveolae-mediated uptake using nystatin or by BSA competition significantly enhanced daidzein-BSA-GdDTPA/CyTE777 uptake by tumor cells in vitro. In vivo, perivascular myofibroblasts generated an effective perivascular barrier excluding delivery of BSA-GdDTPA/CyTE777 to tumor cells. The ability to manipulate caveolae-mediated sequestration of albumin by perivascular tumor myofibroblasts allowed us to effectively overcome this tumor-stroma barrier, increasing delivery of daidzein-BSA-GdDTPA/CyTE777 to the tumor cells in tumor xenografts. Thus, both in vitro and in vivo, endocytosis of daidzein-BSA-GdDTPA/CyTE777 by ovarian carcinoma cells was augmented by albumin or by nystatin. In view of the cardinal role of albumin in affecting the availability and pharmacokinetics of drugs, this approach could potentially also facilitate the delivery of therapeutics and contrast media to tumor cells.

Estrogen has a key role in the regulation of skeletal growth and maintenance of bone mass. Recently, we developed peptides having estrogen-like activity as potential estrogen-based new drugs. The aim of the present study was to evaluate the influence of long-term administration of the most efficacious of these peptides, the hexapeptide EMP-1 (VSWFFE), on bone mass and development. EMP-1 was injected daily to ovariectomized (OVX) and intact young, sexually mature female mice for 10 weeks. Whole femora, including the cartilaginous growth plates were analyzed by micro-computed tomography (microCT). We found that peptide EMP-1 restrains bone growth in OVX mice: it inhibited dramatically bone longitudinal growth (40%), and decreased femoral diaphyseal diameter. Peptide EMP-1 had no effect on bone growth in normal mice, and did not influence the OVX-induced bone loss. We then developed a new microCT methodology to evaluate uncalcified and calcified growth plate parameters. In the OVX mice, peptide EMP-1 reduced volume and thickness of the uncalcified growth plate, a possible cause for the inhibition of bone longitudinal growth. Peptide EMP-1 may be used as a lead compound for the development of drugs to treat acromegalic patients.

We reported previously that high concentrations of either estradiol-17beta (E(2)) or dihydrotestosterone (DHT) inhibit growth of human cultured vascular smooth muscle cells (VSMC), mediated by cell membrane receptors and MAP-kinase-kinase activity (MEK). We now tested whether the presence of the opposite gender's dominant sex hormone modifies these effects. We incubated VSMC with various concentrations of E(2) and DHT or protein bound hormones (E(2)-BSA or T-BSA), alone or in various combinations. High concentration of E(2) or E(2)-BSA inhibited VSMC growth and stimulated MEK. In the presence of 3 nM DHT, high concentration of E(2) no longer inhibited (3)[H] thymidine incorporation or increased MEK. Moreover, when high DHT concentration (300 nM) was added to VSMC exposed to high E(2), VSMC growth actually increased without change in MEK. DHT at 300 nM suppressed VSMC growth and increased MEK while 0.3 nM E(2) had only marginal effect on this interaction, and 30 nM E(2) reversed the inhibitory effect of DHT on cell growth. The inhibitory effects of both E(2) and DHT on VSMC cell growth and the stimulation of MEK was apparently mediated by cell membrane receptors, as it persisted when bovine serum albumin (BSA)-bound hormones were used. Further, inhibition of VSMC growth induced by E(2)-BSA was reversed in the presence of T-BSA and vice versa. These results suggest that while female and male sex hormones affect VSMC growth similarly, they interfere in a dose-, hormone- and MEK-dependent manner with each other's effect.

The use of daunomycin against neoplasms is limited due to its severe cardiotoxicity. The cytotoxicity of daunomycin can be minimized by linking it to an affinity tag. Since ovarian cancer cells are sensitive to isoflavone action, we synthesized a daidzein daunomycin conjugate. In MLS human ovarian cancer cells, the conjugate was shown to have a larger cytotoxic effect than daunomycin per se at a low concentration. The conjugate was then tested in vivo in mice carrying MLS xenografts. Tumour growth in the groups of conjugate and daunomycin was inhibited by >50% as compared to vehicle treated mice. In contrast to daunomycin treated mice, no weight reduction or death was seen in mice treated with the conjugate. In vivo imaging of the fluorescence signal generated by daunomycin indicated uptake of both conjugate and daunomycin by the tumour. Tumour fluorescence was, however, higher in the conjugate treated mice than in the daunomycin treated mice, thus suggesting specific delivery of the drug to the tumour. Histological examination of myocardial tissue indicated that only the daunomycin, but not conjugate treated mice showed cardiac damage. These results indicate that targeting of daunomycin via carboxymethyldaidzein retains daunomycin's cytotoxic effects while averting its toxicity in an ovarian xenograft.

The isoflavones biochanin A ( 1a), genistein ( 1b), and daidzein ( 4) at concentrations >20 microM inhibit cell growth of various cancer cell lines. To enhance the antiproliferative activities of these compounds, we synthesized three analogs, 2-[3-carboxy-(6-tert-butoxycarbonylamino)-hexylamino-propyl]-7,5-dihydroxy-4'-methoxyisoflavone ( 3a), 2-[3-[N-[6-(tert-butoxycarbonyl)-aminohexyl]]-caboxamidopropyl]-5,7,4'-trihydroxyisoflavone ( 3b), and 5-{2-[3-(4-hydroxy-phenyl)-4-oxo-4 H-chromen-7-yloxy]-acetylamino}-pentyl)-carbamic acid tert-butyl ester ( 6). When cancer cells expressing predominantly estrogen receptor mRNA of the beta- relative to alpha-subtype were treated with 3a, 3b, or 6, DNA synthesis was inhibited in a dose-dependent manner, ranging from 15 to 3000 nmol/L, with little inhibitory effect in normal vascular smooth muscle cells. Compound 6 was the most potent one, and its antiproliferative effect in cancer cells was modulated by estrogen and by the apoptosis inhibitor Z-VADFK. When tested in vivo, compound 6 decreased tumor volume of ovarian xenografts by 50%, with no apparent toxicity. Compound 6 may be a promising agent for therapy of cancer either alone or in combination with chemotherapeutic agents.

Human osteoblasts (hOB) produce and respond to 1,25(OH)(2)D(3) (1,25D), suggesting an autocrine/paracrine system. We therefore examined hormonal modulation of the expression and activity of 25 hydroxy-vitamin D(3)-1alpha hydroxylase (1-Ohase) in hOB. Cells from pre- and post-menopausal women or men, were treated with estrogenic compounds and 1-OHase expression and activity were measured. 1-OHase mRNA expression was highest in pre-menopausal women hOB and was increased by all hormones tested. In post-menopausal hOB all hormones except biochainin A (BA) and genistein (G) increased 1-OHase mRNA expressions to less extent. In male-derived hOB only dihydrotestosterone (DHT) and carboxy BA (cBA) increased 1-OHase mRNA expression. 1,25D production from 25(OH)D(3) had a K(m) of approximately 769-400 ng/ml (1.92-1.07 microM) and V(max) of 31.3-17.4 ng/ml (0.078-0.044 microM/60 min/5 x 10(6)cells) respectively, and was increased by all hormones except raloxifene (Ral) with higher stimulation in pre- than in post-menopausal cells. Only BA was almost five times more potent in pre- rather than post-menopausal hOBs. In male hOB only DHT and cBA increased 1,25D production whereas estradiol-17beta (E(2)) had no effect and BA decreased it. These results provide evidence for the expression of 1-OHase mRNA and production of 1,25D in hOBs, which are age and sex dependent and are hormonally modulated. The role of this local autocrine/paracrine 1,25D system in bone physiology deserves further investigation.

The current study sets out to characterize the intracellular localization of the platelet-type 12S-lipoxygenase (12-LO), an enzyme involved in angiotensin-II induced signaling in vascular smooth muscle cells (VSMC). Immunohistochemical analysis of VSMC in vitro or human umbilical arteries in vivo showed a clear cytoplasmic localization. On immunogold electron microscopy, 12-LO was found primarily associated with cytoplasmic VSMC muscle fibrils. Upon angiotensin-II treatment of cultured VSMC, immunoprecipitated 12-LO was found bound to alpha-actin, a component of the cytoplasmic myofilaments. 12-LO/alpha-actin binding was blocked by VSMC pretreatment with the 12-LO inhibitors, baicalien or esculetine and the protein synthesis inhibitor, cycloheximide. Moreover, the binding of 12-LO to alpha-actin was not associated with 12-LO serine or tyrosine phosphorylation. These observations suggest a previously unrecognized angiotensin-II dependent protein interaction in VSMC through which 12-LO protein may be trafficked, for yet undiscovered purposes towards the much more abundantly expressed cytoskeletal protein alpha-actin.

Background: Time-resolved fluorescence immunoassays (TR-FIAs) for phytoestrogens in biological samples are an alternative to mass spectrometric methods. These immunoassays were used to test urine and plasma samples from individuals in a dietary intervention trial aimed at determining the efficacy of dietary isoflavones in reducing the risk of coronary heart disease in postmenopausal women., Methods: We established murine monoclonal TR-FIA methods for daidzein, genistein, and equol. These assays could be performed manually or adapted to an automated analyzer for high throughput and increased accuracy. Analysis of urine was conducted on nonextracted samples. Blood analysis was performed on nonextracted samples for daidzein, whereas genistein and equol required diethyl-ether extraction., Results: Comparison of monoclonal TR-FIA, commercial polyclonal antibody-based TR-FIA, and gas chromatography-mass spectrometry showed correlations (r, 0.911-0.994) across the concentration range observed in the Isoheart study (50 mg/day isoflavones). The concentrations of urinary daidzein and genistein observed during intervention demonstrated good compliance, and a corresponding increase in serum daidzein and genistein confirmed bioavailability of the isoflavone-rich foods; 33 of the 117 volunteers (28.2%) were classified as equol producers on the basis of their urinary equol concentration (>936 nmol/L), and significant differences in the numbers of equol producers were observed between Berlin and the 3 other European cohorts studied., Conclusions: The validated monoclonal TR-FIA methods are applicable for use in large-scale human phytoestrogen intervention studies and can be used to monitor compliance, demonstrate bioavailability, and assess equol producer status.

We have reported previously, that female-derived bone cells responded to 17beta-estradiol (E(2)) and to raloxifene (Ral), whereas male-derived cells responded only to dihydrotestosterone (DHT) when the stimulation of creatine kinase specific activity (CK), which is a marker for hormone responsiveness, was measured. In cells derived from pre-menopausal women, E(2), G, D and Ral stimulated CK to higher extent compared to post-menopausal bone cells, whereas quecertin (Qu), carboxy-biochainin A (cBA) and carboxy-genistein (cG) stimulated CK in both age groups similarly, and biochainin A (BA) stimulated post-menopausal cells to a bit higher extent than pre-menopausal cells. Since the skeletal protective effects of estrogens are not discernable in diabetic women, we tested in this study, the effects of high glucose concentration in the growth medium, on the effects of estrogenic compounds on CK in human-derived bone cells (hObs). Female-derived hObs were grown either in normal (4.5 g/l; 22 mM, NG) or high glucose (9.0 g/l; 44 mM, HG) for 7 days. HG increased constitutive CK, but attenuated E(2)- and DHT-induced CK in female or male hObs, respectively. HG also inhibited genistein (G) and daidzein (D) stimulated CK in female hObs, but not the effects of biochainin A (BA), quecertin (Qu) or Ral. Intracellular, mainly nuclear binding of (3)[H]E(2) was characteristic of the different phytoestrogens in female hObs, was abolished by HG. Membranal binding of Eu-Ov-E(2), was displaced only by E(2)-Ov, ICI, cG-Ov or cD-Ov but decreased total binding of Eu-Ov-E(2) in both age groups and completely abolished the competition with E(2)-Ov or ICI in both age groups, but the competition with cD-Ov and cG-Ov was decreased only slightly but not statistically significant. HG also abolished Eu-BSA-T, which bound similarly male-derived hObs. All hObs expressed mRNA for ERalpha and ERbeta with higher abundance of ERalpha. HG increased mRNA for both ERs in female-derived hObs, but decreased mRNA for both ERs in male-derived hObs. Hence, human bone cells, which express specific nuclear and membranal binding sites for estrogenic compounds, are modulated by HG, leading to altered hormonal responsiveness, which might block important effects of estrogenic compounds, contributing probably to their decreased skeletal preserving properties under hyperglycemia.

We have reported previously, that female-derived bone cells responded to 17beta-estradiol (E(2)) and raloxifene (Ral), and male-derived cells responded only to dihydrotestosterone (DHT) when the stimulation of creatine kinase specific activity (CK), which is a marker for hormone responsiveness, was measured. We also found that pre-treatment with the less-calcemic analog of Vitamin D, JK 1624 F(2)-2 (JKF), upregulated the response of CK to E(2) and Ral. In this study, we analyzed the response of human bone cells from pre- and post-menopausal females and males, to phytoestrogens. Bone cells derived from pre-menopausal women showed greater stimulation of CK than cells from post-menopausal women, after treatment with E(2) (30 nM), daidzein (D, 3000 nM), genistein (G, 3000 nM) and Ral (3000 nM); whereas the responses to biochainin A (BA 3000 nM), quecertin (Qu 3000 nM) or the carboxy derivative of G (cG 300 nM) were not age-dependent. Male-derived cells did not respond to phytoestrogens. When cells derived from female bones at both age groups were pre-treated with JKF, by daily addition of 1nM, for 3 days, there was an upregulation of the response to E(2), Ral, G and D but not to BA or Qu. Nuclear binding of (3)[H] E(2) was characteristic of the different phytoestrogens, with increase of the specific binding after pre-treatment with JKF. In contrast, the membranal binding of E(2)-Ov-Eu, which was specific for the estrogenic compounds except Ral, was inhibited by pre-treatment with JKF except for ICI 161480 (ICI). Male bone cells did not bind E(2)-Ov-Eu but bound T-BSA-Eu; this binding was abolished by pre-treatment with JKF. Pre-treatment with JKF increased mRNA for ERalpha and decreased mRNA for ERbeta in bone cells from both age groups of females and from males, all of which expressed both ERs, with a ratio of ERalpha to ERbeta of 121:1 in pre- and 78:1 in post-menopausal and 105:1 in male bone cells. This study raises the possibility of combined Vitamin D analog and phytoestrogen for hormonal replacement therapy to prevent post-menopausal osteoporosis, which is a subject of ongoing research in animal models.

Estradiol17beta (E2) and the phytoestrogens genistein (G), and daidzein (D) increase creatine kinase (CK) specific activity in primary cell cultures of human female to a greater extent in cells from pre-menopausal than post-menopausal women. Pretreatment with the non-calcemic analog of Vitamin D, JK 1624 F2-2 (JKF), upregulated this estrogenic response at all ages. In contrast, biochainin A (BA) and quercertin (Qu) increased CK with no age dependence or modulation by JKF pretreatment. Both ERalpha and ERbeta present in the cells were upregulated by pretreatment with JKF, as measured by Western blot analysis. Real time PCR showed no significant change in ERalpha mRNA but a marked decrease in ERbeta mRNA in both age groups after JKF treatment. Cells from both age groups had surface binding sites for E2, shown by assays using cell impermeable Europium labeled ovalbumin-E2 conjugate (Eu-Ov-E2). Binding of [3H]-E2 to intracellular E2 receptors (ERs) was similar in both age groups with differences in phytoestrogenic competition. JKF pretreatment increased nuclear but decreased membranal binding in both age groups. These results provide evidence for membranal, in addition to nuclear estrogen receptors which are differentially modulated by a Vitamin D analog.

The present study was designed to determine whether some of the effects of estrogen on human vascular cell growth are exerted through membrane-binding sites, using native as well as novel protein-bound, membrane non-permeant estrogenic complexes. We measured changes in DNA synthesis and creatine kinase-specific activity (CK), after treatment with estradiol-17beta (E(2)), estradiol-17beta-6-(O)-carboxymethyl oxime conjugated to bovine serum albumin (BSA) (E(2)-BSA), 6-carboxymethyl genistein (CG) or 6- carboxymethyl genistein bound to the high molecular protein keyhole limpet hemocyanin (CG-KLH), and 7-(O)-carboxymethyl daidzein (CD) or 7-(O)-carboxymethyl daidzein linked to keyhole limpet hemocyanin (CD-KLH). High concentrations of either E(2) or E(2)-BSA inhibited DNA synthesis in vascular smooth muscle cells (VSMC) (-39% +/- 28% v -32% +/- 15%). Estradiol as well as CG and CD increased DNA synthesis dose dependently in endothelial ECV-304 cells. The CG and CD, as well as CG-KLH and CD-KLH, stimulated DNA synthesis dose dependently in VSMC (66% +/- 2%, 100% +/- 12%, 66% +/- 6%, and 41% +/- 8% at 300 nmol/L, respectively). In contrast all forms of protein-bound hormones were unable to affect DNA synthesis in ECV-304 cells or CK in either cell type. In VSMC, both free and bound hormones increased mitogen-activated protein-kinase (MAPK)-kinase activity, which was blocked by UO126, an inhibitor of MAPK-kinase. Furthermore, the effects of E(2), E(2)-BSA, or CG-KLH on DNA synthesis were inhibited by UO126. Using the E(2)-BSA linked to the fluorescent dye Cy3.5, we directly demonstrated the presence of membrane-binding sites for E(2) in VSMC and ECV 304 cells. Hence, the effects of E(2) on DNA synthesis in human VSMC, but not in endothelial cells, are apparently exerted by membrane-binding sites for E(2) and do not require intracellular entry of E(2) through the classic nuclear receptor route.

In cultured human vascular smooth muscle cells (VSMC), estradiol-17beta (E2) induced a biphasic effect on DNA synthesis, i.e., stimulation at low concentrations and inhibition at high concentrations. Additionally, E2 increased the specific activity of creatine kinase (CK) in these cells. Observations that novel protein-bound membrane impermeant estrogenic complexes could elicit inhibition of DNA synthesis, suggested interaction via membranal binding sites. Nevertheless other effects, such as increasing CK activity were only seen with native E2 but not with E2-BSA, thus indicating that the classical nuclear receptor pathway was involved. In the present report, we confirm that human VSMC express both ERalpha and ERbeta. Further, pretreatment of cultured VSMC with the Vitamin D non-calcemic analog JK 1624 F2-2 (JKF) increased ERalpha mRNA (100-200%) but decreased ERbeta mRNA (30-40%) expression as measured by real time PCR. ERalpha protein expression assessed by Western blot analysis increased (25-50%) in parallel, whereas ERbeta protein expression declines (25-55%). Using ovalbumin bound to E2 (Ov-E2) linked to Eu (Eu-Ov-E2), to assess specific membrane binding sites, we observed that membranal binding was down regulated by JKF by 70-80%. In contrast, total cell binding of 3[H] E2, that nearly entirely represents intracellular E2 binding, was increased by 60-100% by the same Vitamin D analog. The results provide evidence that the effects of JKF on ERalpha/ERbeta as well as on membranal versus nuclear binding of estrogen are divergent and show differential modulation.

Because diabetic women appear not to be protected by estrogen in terms of propensity to cardiovascular disease, we tested the possibility that chronic hyperglycemia modulates the effects of E(2) on vascular cell growth in vitro. Human endothelial cells (E304) and vascular smooth muscle cells (VSMC) were grown in normal glucose (5.5 mmol/l), high glucose (22 mmol/l) or high manitol (22 nmol/l; an osmotic control) for 7 days. In endothelial cells glucose per se stimulated DNA synthesis. However E(2)- (but not RAL-) stimulated [3H] thymidine incorporation was attenuated in the presence of high glucose. In parallel, E(2)-dependent MAP-kinase-kinase activity was blocked in the presence of high glucose. High glucose increased basal creatine kinase (CK) specific activity, but E(2)-stimulated CK was not significantly impaired in the presence of high glucose. In VSMC, high glucose prevented the inhibitory effect of high E(2) (but not of high RAL) concentrations on DNA synthesis. High glucose also prevented E(2)-induced MAP-kinase-kinase activity. In contrast, while high glucose augmented basal CK, the relative E(2)-induced changes were roughly equal in normal and high high glucose media. Hence, high glucose blocks several effects of E(2) on vascular cell growth, which are mediated, in part, via the MAP-kinase system and are likely contributors to E(2)'s anti-atherosclerotic properties. Since RAL's estrogen-mimetic effects on human vascular cell growth were independent of MAP-kinase activation and were not affected by hyperglycemia, the potential use of RAL to circumvent the loss of estrogen function induced by hyperglycemia and diabetes in the human vasculature should be further explored.

Currently used antiestrogenic drugs against hormone-dependent breast cancer, and estrogenic drugs used in treatment of osteoporosis, are associated with risk factors. Therefore, there is a strong need to develop selective estrogen receptor modulators with better tissue selectivity. In a recent study (Peptides, 2002, Vol. 3, 573-580), we used a monoclonal antibody to estradiol (mAb-E2) to screen a phage-display peptide library. We identified a 15-mer peptide (peptide H5) that recognizes mAb-E2 (IC(50) 1 microM) and estrogen receptor (ER)alpha (IC(50) 500 microM) but not ERbeta, and displays estrogen-like activity in vitro and in vivo. In this study, we designed and prepared peptides based on peptide H5, which possess improved estrogenic activity, by evaluating their binding to mAb-E2 and to ERs. Initially, we determined the minimal binding sequence of peptide H5 capable of binding mAb-E2 and ER. Subsequently, systematic single-residue replacements of the minimal sequence, followed by multiple-residue replacements, yielded hexa- and heptapeptides with increased affinities to mAb-E2 and to ER. The most promising peptides, VSWFFE (EMP-1) and VSWFFED (EMP-2) (EMP: estrogen-mimetic peptide), bind mAb-E2 with high affinity (IC(50) of 6 and 30 nM, respectively), recognize ERs with increased affinity (IC(50) of 100 microM for ERalpha, and 100-250 microM for ERbeta), and possess estrogenic activity in vivo. The short peptides described in this study may be used as potential lead compounds for developing new ER ligands., ((c) 2004 Wiley Periodicals, Inc.)

Background: Monitoring of reproductive steroid hormones at the population level requires frequent measurements, hormones or metabolites that remain stable under less than ideal collection and storage conditions, a long-term supply of antibodies, and assays useful for a range of populations. We developed enzyme immunoassays for urinary pregnanediol 3-glucuronide (PDG) and estrone conjugates (E1Cs) that meet these criteria., Methods: Enzyme immunoassays based on monoclonal antibodies were evaluated for specificity, detection limit, parallelism, recovery, and imprecision. Paired urine and serum specimens were analyzed throughout menstrual cycles of 30 US women. Assay application in different populations was examined with 23 US and 42 Bangladeshi specimens. Metabolite stability in urine was evaluated for 0-8 days at room temperature and for 0-10 freeze-thaw cycles., Results: Recoveries were 108% for the PDG assay and 105% for the E1C assay. Serially diluted specimens exhibited parallelism with calibration curves in both assays. Inter- and intraassay CVs were <11%. Urinary and serum concentrations were highly correlated: r = 0.93 for E1C-estradiol; r = 0.98 for PDG-progesterone. All Bangladeshi and US specimens were above detection limits (PDG, 21 nmol/L; E1C, 0.27 nmol/L). Bangladeshi women had lower follicular phase PDG and lower luteal phase PDG and E1Cs than US women. Stability experiments showed a maximum decrease in concentration for each metabolite of <4% per day at room temperature and no significant decrease associated with number of freeze-thaw cycles., Conclusions: These enzyme immunoassays can be used for the field conditions and population variation in hormone metabolite concentrations encountered in cross-cultural research.

The predominance of autoimmune diseases among women suggests that estrogen may modulate immune function. Monocytes and macrophages are important in initiating, maintaining, and resolving inflammatory responses through cell-signaling molecules, which control immune cell survival. One important mechanism of cell survival is mediated by the Fas/Fas ligand (FasL) system. In this study, the link between estrogen, monocytes/macrophages, and the Fas/FasL system was investigated. Estrogen treatment increased FasL expression in monocytes through the binding of the estrogen receptors (ER) to the estrogen recognizing elements and AP-1 motifs present at the FasL promoter. Furthermore, estrogen induced apoptosis in monocytes expressing ERbeta, but not in monocyte-differentiated macrophages expressing ERalpha. The expression of either ERalpha or ERbeta and their response to estrogen in monocytes was found to be dependent on the their stage of cell differentiation. Previously, we have shown that estrogen replacement therapy in postmenopausal women decreased the number of circulating monocytes. In this study, we have characterized the molecular mechanism by which estrogen regulates monocytes homeostasis. These findings indicate that estrogen may regulate immune cell survival through the Fas/FasL system. There is biological relevance to these findings in view of studies showing that accumulation of activated monocytes is involved in the pathogenesis of conditions such as vasculititis, arteriosclerosis, and rheumatoid arthritis.

Vascular endothelial growth factor (VEGF) is one of the key growth factors regulating tumor angiogenesis and thus it is one of the primary targets for antiangiogenic therapy. The long-term effects of VEGF include induction of proliferation and migration of endothelial cells, tube formation and maintenance of the immature capillaries. The early effects of VEGF include vasodilation and increased permeability. We hypothesize that the early responses to VEGF can serve to develop a quantitative measure of the activity of VEGF, and therefore may be applicable for monitoring the efficacy of systemic suppression of VEGF signaling during antiangiogenic therapy. For that end we tested the ability of MRI and fluorescence microscopy to detect the early response to intradermal VEGF165 in nude mice. VEGF-induced local vasodilation and increased permeability was detected by intravenous administration of macromolecular biotin-BSA-GdDTPA(23) 30 min after intradermal administration of VEGF. Contrast leak showed saturation kinetics. Delayed contrast administration (90 min after intradermal administration of VEGF) resulted in low contrast leak and demonstrated that the saturation kinetics is not due to contrast equilibration between plasma and the interstitial space, but rather is due to suppression of vascular permeability. Permeability was restored by a second bolus of VEGF, showing that the saturation kinetics is primarily due to inactivation of the growth factor. Confocal microscopy of fluorescent BSA-FITC confirmed the permeability changes monitored by MRI. Moreover, confocal microscopy showed efficient lymphatic uptake of the extravasated contrast material specifically in regions of VEGF induced hyper-permeability., (Copyright 2002 John Wiley & Sons, Ltd.)

We describe a novel approach to develop peptides with estrogen like activity using a monoclonal antibody specific to estradiol (mAb E2-15) for the affinity selection of phage displayed peptides from a combinatorial peptide library. Based on the sequences of the selected phage, we synthesized a 15-mer linear peptide LPALDPTKRWFFETK which was derivatized to a 23 mer cyclic peptide CAELPALDPTKRWFFETKPPPPC. Both peptides displayed estrogen-like activity according to the following criteria:(i) in inhibiting the binding of [3H]estradiol to mAb E2-15 and to estrogen receptor (ER)alpha; (ii) in inducing transcriptional activity in MCF7 human breast cancer cells transfected with an estrogen receptor element luciferase construct and (iii) in causing an increase in creatine kinase specific activity in rat tissues in vivo. This approach can be employed to design peptide mimetic for other hormones as well.

We investigated the possibility of labeling two biologically active peptides, epidermal growth factor (EGF) and neurotensin (NT), with europium (Eu)-diethylenetriaminepentaacetic acid. More specifically, we tested them as probes in studying receptor binding using time-resolved fluorescence of Eu3+. The relatively simple synthesis yields ligands with acceptable binding characteristics similar to isotopically labeled derivatives. The binding affinity (Kd) of labeled Eu-EGF to human A431 epidermal carcinoid cells was 3.6 +/- 1.2 nM, similar to the reported Kd values of EGF, whereas the Kd of Eu-NT to human HT29 colon cancer cells (7.4 +/- 0.5 nM) or to Chinese hamster ovary (CHO) cells transfected with the high-affinity NT receptor (CHO-NT1) were about 10-fold higher than the Kd values of NT. The bioactivity of the Eu-labeled EGF as determined by stimulation of cultured murine D1 hematopoietic cell proliferation was nearly the same as that obtained with native EGF. The maximal stimulation of Ca2+ influx with NT and Eu-NT in CHO-NT1 cells was similar, but the respective K0.5 values were 20 pM and 1 nM, corresponding to differences in the binding affinities previously described. The results of these studies indicate that Eu labeling of peptide hormones and growth factor molecules ranging from 10(3) to 10(5) Da can be conveniently accomplished. Importantly, the Eu-labeled products are stable for approximately 2 years and are completely safe for laboratory use compared to the biohazardous radioligands. Thus, Eu-labeled peptides present an attractive alternative for commonly used radiolabeled ligands in biological studies in general and in receptor assays in particular.

Background: The aim of this study was to assess the effect of phytoestrogens on the human vascular wall in vitro., Methods: We compared the effects of E2 to those of genistein (G), daidzein (D), biochanin A (BA), equol (EQ), and quecertin (Qu) on 3[H] thymidine incorporation and creatine phosphokinase (CK) activity in human vascular smooth muscle cells (VSMC) and in a human endothelial cell line (E304)., Results: In VSMC, E2, the estrogen antagonist raloxifene (RAL), G, and D stimulated DNA synthesis at low concentrations and suppressed 3[H] thymidine incorporation at higher concentrations. In contrast, BA and EQ had a monophasic stimulatory effect on 3[H] thymidine incorporation (87% +/- 9% and 54% +/- 17%, respectively) whereas Qu had only an inhibitory effect (-36 +/- 16% at 30 nmol/L). In E304 cells, all phytoestrogens stimulated DNA synthesis in a dose-related manner. In both cell types E2, RAL as well as all phytoestrogens increased CK-specific activity. The administration of phytoestrogens to immature female rats resulted in increased CK in the aorta (Ao) (60% to 220%) and in the left ventricle of the heart (Lv) (45% to 160%). Similar increases in Ao and Lv CK were also induced by E2 and all five phytoestrogens in ovariectomized (OVX) female rats. RAL antagonized phytoestrogen-induced CK activity in human vascular cells and in the rat Ao and Lv tissue but did not block phytoestrogen effects on DNA synthesis in human VSMC., Conclusions: Although phytoestrogens have estrogen-mimetic effects on cell growth and CK in cultured human vascular cells and on CK in rat vascular tissues in vivo, the effects on human VSMC replication are highly dependent on the concentration and the particular phytoestrogen under investigation.

The lipoxygenase pathway has been implicated in the growth, migration, and contraction of vascular smooth muscle cells (VSMCs). However, the precise type of lipoxygenase present in the vascular wall has not been characterized. In this study, we used a specific reverse-transcriptase polymerase chain reaction method with 2 sets of specific primers on total RNA and polyA (+)RNA of normal human VSMCs prepared from umbilical artery. Two forms of platelet-type 12-lipoxygenase mRNA were present in human VSMCs: the already published form cloned from human erythroleukemia cells and a variant form of platelet-type 12-lipoxygenase, which includes 2 additional sequences consistent with the 2 introns (D and E). This novel form of 12-lipoxygenase poly A (+)RNA was downregulated by lipopolysaccharide (10 ug/ml) and upregulated by epidermal growth factor (100 ng/ml) but was not affected by angiotensin II (10(-7) mol/l). We developed a rabbit anti-human platelet-type 12-lipoxygenase polyclonal antibody directed against a 24-amino acid peptide encoded within exon 4. Western immunoblotting of protein extracted from VSMCs and umbilical artery and platelet extract with this antibody showed a coordinate 110-kDa protein and the already-described 70-kDa band detected in platelets and cord homogenate. Another 120-kDa protein was consistently detected in cord extracts but not in platelet or VSMC homogenates. The immunohistochemistry study performed with the same antibody showed extensive cytoplasmic staining of VSMCs. The specific role of these different forms of platelet-type 12-lipoxygenase is subject to further investigation.

Problem: Estrogen induces atrophy in the thymus by an unknown mechanism. Since the Fas/FasL system is one of the main pathways in T cell apoptosis, we tested the hypothesis that estrogen-induced thymic atrophy is mediated by the Fas/FasL system., Methods of Study: In vivo experiments were done using ovariectomized female rats treated with estrogen or saline. In vitro experiments were performed using isolated thymocytes. Estrogen receptor (ER) alpha and beta expression was characterized using flow cytometry, RT-PCR and immunofluorescence. Fas and FasL mRNA and protein expression was evaluated using RT-PCR and Western blot analysis respectively., Results: ERalpha and ERbeta are present in thymocytes and stromal cells. ER expression is mainly localized in the Double Positive CD4+CD8+ thymocytes. Estrogen treatment decreases thymus size and increase FasL expression., Conclusion: CD4+CD8+ thymocytes and thymic stroma cells express ERalpha and ERbeta. In vivo and in vitro we showed that estrogen treatment increases FasL expression while decreasing thymus cell number. These findings support the hypothesis that estrogen-induced thymic atrophy occurs as a result of apoptosis and is mediated by estrogen-induced FasL expression.

We describe here a strategy for photodynamic eradication of solid melanoma tumors that is based on photo-induced vascular destruction. The suggested protocol relies on synchronizing illumination with maximal circulating drug concentration in the tumor vasculature attained within the first minute after administrating the sensitizer. This differs from conventional photodynamic therapy (PDT) of tumors where illumination coincides with a maximal concentration differential of sensitizer in favor of the tumor, relative to the normal surrounding tissue. This time window is often achieved after a delay (3-48 h) following sensitizer administration. We used a novel photosensitizer, bacteriochlorophyll-serine (Bchl-Ser), which is water soluble, highly toxic upon illumination in the near-infrared (lambda max 765-780 nm) and clears from the circulation in less than 24 h. Nude CD1 mice bearing malignant M2R melanotic melanoma xenografts (76-212 mm3) received a single complete treatment session. Massive vascular damage was already apparent 1 h after treatment. Changes in vascular permeability were observed in vivo using contrast-enhanced magnetic resonance imaging (MRI), with the contrast reagent Gd-DTPA, by shortening spin-spin relaxation time because of hemorrhage formation and by determination of vascular macromolecular leakage. Twenty-four hours after treatment a complete arrest of vascular perfusion was observed by Gd-DTPA-enhanced MRI. Histopathology performed at the same time confirmed primary vascular damage with occlusive thrombi, hemorrhage and tumor necrosis. The success rate of cure of over 80% with Bchl-Ser indicates the benefits of the short and effective treatment protocol. Combining the sensitizer administration and illumination steps into one treatment session (30 min) suggests a clear advantage for future PDT of solid tumors.

Epidermal growth factor (EGF) receptor (ErbB1)-associated tyrosine kinase inhibitors may act as potential chemotherapeutic agents. In order to assess the inhibitory activity of these compounds, we developed a simple and sensitive assay based on time-resolved fluorescence. In this technique, crude cell lysates bearing the ErbB1 receptor were captured in microtitre plates immobilized with monoclonal anti-ErbB1 antibody SG 565. Subsequently, the phosphotyrosine content of the cell lysates was quantified by a europium-labelled anti-phosphotyrosine antibody. Thus, genistein, a tyrosine kinase inhibitor, was capable of reducing by half the tyrosine phosphorylation caused by the binding of EGF to A431 cells, whereas 6-carboxymethyl genistein did not inhibit protein tyrosine phosphorylation. This assay is simple to perform, does not use radioactive substrates, and can be useful for screening EGF receptor tyrosine kinase inhibitors from natural products or synthetic compounds. Moreover, the assay has a high signal:noise ratio and is suitable for large-scale screening., (Copyright 2000 John Wiley & Sons, Ltd.)

Background: Estrogen receptor (ER) levels in tumor cells are important for determining the outcome of treatment and the prognosis of breast cancer patients. Flow cytometry is a convenient tool for quantifying the ER in cells, but a more sensitive, reproducible method for immunostaining the ER with anti-ER antibody is needed. Materials and Methods ER-positive human breast cancer cells MCF-7 and T47D, and ER-negative MDA-MBA-321 cells, were fixed and permeabilized by three different protocols. The cells were then stained by indirect immunofluorescence, using two commercial antibodies to ER (MA1-310 and DAKO 1D5), or by direct immunofluorescence using FITC-labeled anti-idiotypic antibody clone 1D(5). The stained cells were analyzed by flow cytometry., Results: The fixation of cells with a mixture of 0.25% paraformaldehyde and 70% methanol, permeabilization with 0.05% Triton X-100, and increasing antibody and antigen reaction time led to 80-99% of cells being stained with anti-ER antibodies. The relative brightness of ER immunostaining was as follows: anti-idiotypic antibody ID5 > MA1-310 > DAKO 1D5., Conclusions: Direct immunofluorescence with the FITC-labeled anti-idiotypic antibody of permeabilized cells resulted in improved specific staining of the ER, as compared to indirect immunofluorescence with anti-ER antibodies of fixed and permeabilized cells. Increasing the length of staining, and treatment of cells with Triton X-100, are both necessary to improve the staining of intracellular antigen for flow cytometric analysis., (Copyright 2000 Wiley-Liss, Inc.)

4-chloro-androst-4-ene-3,17-dione (CLAD) and 4-chlorotestosterone (clostebol, beta-CLT or CLT) were made immunogenic by coupling to protein carriers via the 3 and 17 positions, respectively. These immunogens were used to elicit polyclonal and monoclonal antibodies to CLAD and to clostebol. The antibodies were characterized in an enzyme immunoassay for sensitivity and specificity. Polyclonal antisera generated through position 17 reacted preferentially with 4-chlorotestosterone-17-acetate (clostebol acetate, CLTA), 4-chloro-epitestosterone (epi-clostebol, 17alpha-clostebol, 17alpha-CLT), and clostebol, whereas polyclonal antisera generated through the 3 position almost did not react with these derivatives. Interestingly, the monoclonal antibody generated through the 3 position recognized (35%) epi-clostebol. These results suggest that polyclonal antisera generated through the 17 position have a broad specificity profile and can be used to analyze by immunoassay methods urinary metabolites of clostebol acetate and thereby detect the illegal use of clostebol acetate in livestock farming.

Ovarian hormones are biomarkers for breast cancer risk. Soybean consumption may be responsible in part for lower levels of ovarian hormones and decreased rates of breast cancer in women in Asia compared with Western populations. Soybeans contain a significant amount of the isoflavones daidzein and genistein, which are weak estrogens. The purpose of this study was to determine whether soya feeding decreases circulating levels of ovarian hormones and gonadotropins. Ten healthy, regularly cycling women consumed a constant soya-containing diet on a metabolic unit, starting on day 2 of a menstrual cycle until day 2 of the next cycle. Blood and urine samples were obtained daily for one menstrual cycle before and during soy feeding. The diet was calculated to maintain constant body weight, included 400 kilocalories from a 36-ounce portion of soymilk, and provided 113-207 mg/day (154.0+/-8.4 mg/day, mean +/- SE) of total isoflavones. For the group, the soya diet provided more carbohydrate and less protein than the home diets. Daily consumption of the soya diet reduced circulating levels of 17beta-estradiol by 25% (P<0.01, Wilcoxon signed rank test, two-tailed) and of progesterone by 45% (P<0.0001) compared with levels during the home diet period but had no effect on luteinizing hormone or follicle-stimulating hormone. Mean menstrual cycle length did not change during the soya diet; a slight decrease in mean luteal cycle length was marginally statistically significant (P = 0.06). Urinary excretion of isoflavones was 33.8+/-5.3 mg/day (mean +/- SE) and when expressed as percentage of intake, varied substantially (21.9+/-3.3% of intake; range, 9.1-36.7%) among the subjects. Mean daily serum levels of daidzein and genistein (free and conjugated forms) 15 h after soymilk were 2.89+/-0.53 microg/ml and 0.85+/-0.22 microg/ml, respectively, indicating systemic bioavailability of these substances. Secondary analyses by multiple regression showed that decreases in follicular and luteal phase 17beta-estradiol levels were positively associated with urinary isoflavone excretion, an association affected by age, and were inversely associated with decreases in protein intake. Decreases in progesterone levels during the soya diet were inversely associated with increases in intakes of genistein and were affected by the interaction of the intakes of daidzein with energy or with fiber. Consumption of an isoflavone-containing soya diet reduced levels of ovarian steroids in normal women over the entire menstrual cycle without affecting gonadotropins. This suggests that at least under the conditions of this study, soya-induced reductions of circulating ovarian steroids are not mediated by gonadotropins. Decreases in ovarian hormones are related to isoflavones contained in soy and also to energy intake and other components such as protein and fiber but not fat. Our results may explain decreased ovarian hormone levels and decreased risk of breast cancer in populations consuming soya diets and have implications for reducing breast cancer risk by dietary intervention.

During neoplastic growth and metastasis, the immune system responds to the tumor by developing both cellular and humoral immune responses. In spite of this active response, tumor cells escape immune surveillance. We previously showed that FasL expression by breast tumor plays a central role in the induction of apoptosis of infiltrating Fas-immune cells providing the mechanism for tumor immune privilege. In the present study, we showed that FasL in breast tissue is functionally active, and estrogen and tamoxifen regulate its expression. We identified an estrogen recognizing element like-motif in the promoter region of the FasL gene, suggesting direct estrogen effects on FasL expression. This was confirmed by an increase in FasL expression in both RNA and protein levels in hormone sensitive breast cancer cells treated with estradiol. This effect is receptor mediated since tamoxifen blocked the estrogenic effect. Interestingly, tamoxifen also inhibited FasL expression in estrogen-depleted conditions. Moreover, an increase in FasL in breast cancer cells induces apoptosis in Fas bearing T cells and, tamoxifen blocks the induction of apoptosis. These studies provide evidence that tamoxifen inhibits FasL expression, allowing the killing of cancer cells by activated lymphocytes. This partially explains the protective effect of tamoxifen against breast cancer.

We have previously reported that estradiol (E2) and dihydrotestosterone (DHT) regulate cell growth in human umbilical arterial smooth muscle cells (SMC) and in an endothelial cell line (E304). In SMC both gonadal steroids stimulated DNA synthesis at low concentrations and suppressed 3[H] thymidine incorporation at high concentrations, whereas in E304 cells E2 and DHT dose dependently enhanced DNA synthesis. In both cell types gonadal steroids also induced the specific activity of creatine kinase BB (CK). Previous evidence suggets that the in vitro and in vivo CK responses to gonadal steroids in bone cells are upregulated by pretreatment with vitamin D analogs due to increased level of cellular estrogen receptors (ER). Here we analyzed the interaction of the vitamin D analogs hexafluorovitamin D (FL), JK-1624 F2-2 (JKF), and CB 1093 (CB) with gonadal steroids in regulating DNA synthesis and CK activity in human vascular cells in vitro. In E304 cells, daily treatment with FL, JKF, or CB (1 nmol/L for 3 days) increased DNA synthesis by 110 +/- 11%, 65 +/- 16%, and 88 +/- 23% respectively. In contrast, the same analogs inhibited 3[H] thymidine incorporation by 52 +/- 21%, 46 +/- 19%, and 50 +/- 10%, respectively, in SMC. In both cell types all three analogs increased CK by 25% to 75% and amplified the CK response to E2 and to DHT by twofold to threefold. In E304 cells the vitamin D analogs also increased DNA response to gonadal steroids from 50% to 60% to 200% to 280%. In SMC these analogs did not modify the DNA synthetic response to a low E2 concentration, but prevented the suppression of DNA synthesis exerted by high concentrations of E2 and DHT. Vitamin D inhibitors known to block cellular calcium mobilization, had no effect on the proliferative activity induced by vitamin D analogs. However, the inhibitor of the nuclear effects of vitamin D, ZK 159222, blocked the stimulatory effects of CB on DNA synthesis in E304 cells. Finally, both 1,25(OH)2 D3, and JKF decreased the expression of ERbeta proteins in SMC and increased the ERalpha isoform in E304 cells by 40% to 75%. The results indicate that vascular cells are targets for both vitamin D and gonadal steroid action and suggest a possible interaction between these hormones in the regulation of cell proliferation via modulation of vascular ER or interaction with proteins associated with ER.

Different types of lymphocytes have different roles in tumor suppression. Thus, their expression of apoptosis-related proteins (ARP - Fas and Fas ligand, bcl-2, p53) in lymphocytes and their apoptosis were analyzed immunohistochemically in ovarian tumors of different grades. Ovaries without oncologic disorders had few lymphocytes, mainly T cells, and no ARP. Benign cysts presented features of weak immune reaction: small lymphoid infiltration and few lymphocytes. The ARP were present in 13.7% to 23.5% of the lymphocytes, and apoptosis was rare. In borderline tumors, expansion of lymphoid infiltrates and increased density of lymphocytes resulted in a tenfold rise in total lymphocytes, reflecting intensification of the immune response. Most lymphocytes were T cells (92%) predominated by CD8+ cells that were in direct contact with tumor epithelial cells. ARP species were found in 47% to 65% of the lymphocytes, and apoptosis in 2.2%. In carcinomas with ligh lymphoid infiltration, lymphocytes were 2.5 times more abundant, and the apoptotic index as well as the number of CD20+ and CD25+ lymphocytes rose sharply, whereas bcl-2 positive lymphocytes decreased to 8% of their number in borderline tumors. In carcinomas with low lymphoid infiltration, the total lymphocyte count decreased eightfold compared to carcinomas with high lymphoid infiltration, reflecting the deep subcompensation of the lymphoid system. Few p53-positive lymphocytes were found in the carcinomas. In conclusion, we found a positive correlation between apoptosis and the numbers of CD4+ or CD8+ lymphocytes in epithelial ovarian tumors. This correlation could reflect the antitumor activity of T cells. However, the high expression of ARP studied by immune cells at the vicinity of the tumor ARP reveals the lymphoid vulnerability to apoptosis, resulting in devastation of the lymphoid tissue, and consequently in tumor progression.

An immunoaffinity chromatographic (IAC) method for isolating sulfamethazine (SMZ) from incurred urine samples was developed. This was achieved by (i) generating polyclonal antibodies that recognize equally well SMZ and its major urinary metabolites, (ii) evaluating in an ELISA procedure the influence of methanol, salt and pH on the antigen-antibody interaction in order to determine the optimum conditions for IAC and (iii) covalent coupling of the IgG fractions of anti-SMZ to CNBr activated Sepharose for the preparation of re-usable immunoaffinity columns, having a high capacity for SMZ (1900 ng SMZ mL-1 gel). For desorbing SMZ from the immunoaffinity column, different elution modes were evaluated, with 40% MeOH-0.1 mol L-1 HOAc-0.5 mol L-1 NaCl being the most efficient combination. Using the IAC column for processing SMZ spiked urine samples resulted in high recoveries, ranging from 92 to 100%. Because of the high cross-reactivity with the major metabolites of SMZ present in urine of treated animals, the antibodies show excellent properties for use in both IAC and ELISA. For the isolation and concentration of the parent drug and its major metabolites, the urine could be applied directly to the IAC column, without the time-consuming step of deconjugation. Moreover, the use of IAC prior to ELISA for the analysis of incurred urine samples showed good efficiency for the elimination of matrix interferences. Owing to the urine-tissue relationship, the urine concentrations can be used to predict the presence of the parent drug in tissues and so possible violations of the maximum residue limit (MRL) can be controlled.

Platelet-derived endothelial cell growth factor (PD-ECGF) is linked to angiogenesis in human cancer. Direct studies have demonstrated that PD-ECGF is a potent mitogen for endothelial cells in vivo. Because endothelial repair and smooth muscle cell proliferation are two processes that affect arterial wall structure and tone, we analyzed the effects of PD-ECGF on DNA synthesis and creatine kinase BB-specific activity (CK) in human umbilical artery smooth muscle cells (SMC) and in a human umbilical endothelial cell line (E304). In SMC, PD-ECGF (0.001 to 10 U/mL) inhibited DNA synthesis dose dependently (-24% + 6% to -63% + 15%) assessed by 3[H]thymidine incorporation into DNA, whereas in E304 it stimulated DNA synthesis dose dependently (30% + 4% to 100% + 4%). In both SMC and E304, however, PD-ECGF elicited an increase in CK-specific activity by 54% to 130% and 79% to 163%, respectively. These effects were reversed by a specific anti-PD-ECGF antibody. In E304 cells PD-ECGF enhanced 17beta-estradiol (E2) or dihydrotestosterone (DHT)-induced DNA synthesis from 56% to 122% and from 127% to 359%, and CK activity from 70% to 180% and from 90% to 190%, respectively. In SMC PD-ECGF, an inhibitor of 3[H]thymidine incorporation by itself, markedly enhanced the stimulatory effect of low concentrations of E2 and DHT on 3[H]thymidine incorporation. It also increased E2 and DHT CK induction from 40% to 140% and from 52% to 120%, respectively. In both E304 and SMC, PD-ECGF inhibited the proliferative and the CK-inducing effects of platelet-derived growth factor (PDGF) and immunoglobulin F1 (IGF1). Thus, PD-ECGF, an established growth promoter for endothelial cells, is a potent inhibitor of DNA synthesis in human arterial SMC. However, in both E304 endothelial cells and SMC, PD-ECGF enhances the stimulatory effect of low concentrations of gonadal steroids on 3[H]thymidine incorporation. PD-ECGF antagonizes PDGF- and IGF1-induced DNA synthesis in both E304 and SMC cells. By inhibiting arterial SMC proliferation and accelerating endothelial cell replication, PD-ECGF may buffer the effect of PDGF and favorably modulate arterial wall response to injury.