Phase 1 open-label trial of intravenous administration of MVA-BN-brachyury-TRICOM vaccine in patients with advanced cancer.

BACKGROUND: MVA-BN-brachyury-TRICOM is a recombinant vector-based therapeutic cancer vaccine designed to induce an immune response against brachyury. Brachyury, a transcription factor overexpressed in advanced cancers, has been associated with treatment resistance, epithelial-to-mesenchymal transition, and metastatic potential. MVA-BN-brachyury-TRICOM has demonstrated immunogenicity and safety in previous clinical trials of subcutaneously administered vaccine. Preclinical studies have suggested that intravenous administration of therapeutic vaccines can induce superior CD8+ T cell responses, higher levels of systemic cytokine release, and stronger natural killer cell activation and proliferation. This is the first-in-human study of the intravenous administration of MVA-BN-brachyury-TRICOM. METHODS: Between January 2020 and March 2021, 13 patients were treated on a phase 1, open-label, 3+3 design, dose-escalation study at the National Institutes of Health Clinical Center. The study population was adults with advanced solid tumors and was enriched for chordoma, a rare sarcoma of the notochord that overexpresses brachyury. Vaccine was administered intravenously at three DLs on days 1, 22, and 43. Blood samples were taken to assess drug pharmacokinetics and immune activation. Imaging was conducted at baseline, 1 month, and 3 months post-treatment. The primary endpoint was safety and tolerability as determined by the frequency of dose-limiting toxicities; a secondary endpoint was determination of the recommended phase 2 dose. RESULTS: No dose-limiting toxicities were observed and no serious adverse events were attributed to the vaccine. Vaccine-related toxicities were consistent with class profile (ie, influenza-like symptoms). Cytokine release syndrome up to grade 2 was observed with no adverse outcomes. Dose-effect trend was observed for fever, chills/rigor, and hypotension. Efficacy analysis of objective response rate per RECIST 1.1 at the end of study showed one patient with a partial response, four with stable disease, and eight with progressive disease. Three patients with stable disease experienced clinical benefit in the form of improvement in pain. Immune correlatives showed T cell activation against brachyury and other tumor-associated cascade antigens. CONCLUSIONS: Intravenous administration of MVA-BN-brachyury-TRICOM vaccine was safe and tolerable. Maximum tolerated dose was not reached. The maximum administered dose was 109 infectious units every 3 weeks for three doses. This dose was selected as the recommended phase 2 dose. TRIAL REGISTRATION NUMBER: NCT04134312.

The microtubule end-binding protein EB2 is a central regulator of microtubule reorganisation in apico-basal epithelial differentiation.

Microtubule end-binding (EB) proteins influence microtubule dynamic instability, a process that is essential for microtubule reorganisation during apico-basal epithelial differentiation. Here, we establish for the first time that expression of EB2, but not that of EB1, is crucial for initial microtubule reorganisation during apico-basal epithelial differentiation, and that EB2 downregulation promotes bundle formation. EB2 siRNA knockdown during early stages of apico-basal differentiation prevented microtubule reorganisation, whereas its downregulation at later stages promoted microtubule stability and bundle formation. Interestingly, although EB1 is not essential for microtubule reorganisation, its knockdown prevented apico-basal bundle formation and epithelial elongation. siRNA depletion of EB2 in undifferentiated epithelial cells induced the formation of straight, less dynamic microtubules with EB1 and ACF7 lattice association and co-alignment with actin filaments, a phenotype that could be rescued by inhibition with formin. Importantly, in situ inner ear and intestinal crypt epithelial tissue revealed direct correlations between a low level of EB2 expression and the presence of apico-basal microtubule bundles, which were absent where EB2 was elevated. EB2 is evidently important for initial microtubule reorganisation during epithelial polarisation, whereas its downregulation facilitates EB1 and ACF7 microtubule lattice association, microtubule-actin filament co-alignment and bundle formation. The spatiotemporal expression of EB2 thus dramatically influences microtubule organisation, EB1 and ACF7 deployment and epithelial differentiation.

Fetal fibronectin signaling induces matrix metalloproteases and cyclooxygenase-2 (COX-2) in amnion cells and preterm birth in mice.

Fetal fibronectin (fFN) in cervical and vaginal secretions has been used as a predictor of preterm delivery. Here, we clarified the pathological function of fFN on cell type-specific matrix metalloproteinases (MMPs) and prostaglandin synthesis in fetal membranes. Treatment of amnion mesenchymal cells with fFN resulted in dramatic increases in MMP-1 and MMP-9 mRNA and enzymatic activity as well as COX-2 mRNA and prostaglandin E(2) synthesis, activating both NFκB and ERK1/2 signaling. Fetal FN-induced increases in MMPs and COX-2 were mediated through its extra domain A and Toll-like receptor 4 expressed in mesenchymal cells. Lipopolysaccharide and TNF-α increased the release of free FN in medium of amnion epithelial cells in culture. Finally, injection of fFN in pregnant mice resulted in preterm birth. Collectively, these results indicate that fFN is not only a marker of preterm delivery but also plays a significant role in the pathogenesis of preterm labor and premature rupture of fetal membranes.

Long-term engagement of CD6 and ALCAM is essential for T-cell proliferation induced by dendritic cells.

Interactions between T cells and antigen-presenting cells (APCs) are the first step in the induction of an adaptive immune response. Here, we show that CD6 and its ligand activated leukocyte cell adhesion molecule (ALCAM) are actively recruited to the antigen-induced dendritic cell (DC)-T-cell contact zone. Moreover, ALCAM-blocking antibodies interfere with DC-T-cell conjugate formation, demonstrating that CD6-ALCAM binding is essential for stable T-cell-APC contact. We now demonstrate that besides their role in establishing initial contacts, CD6-ALCAM interactions are also required during the proliferative phase of the T-cell response; the presence of CD6-blocking antibodies or recombinant ALCAM-Fc proteins results in a strong and sustained inhibition of T-cell proliferation. Furthermore, simultaneous crosslinking of CD6 and CD3 induces enhanced proliferation and transcriptional activity to a similar level as observed after CD3 and CD28 co-crosslinking, demonstrating that CD6 is an important costimulatory molecule. The stability of ALCAM-CD6 binding, which contrasts with transient homotypic ALCAM-ALCAM interactions, further supports the long-lasting effects observed on T-cell proliferation. Taken together, we demonstrate that CD6 and ALCAM form a key adhesive receptor-ligand pair that is not only involved in early DC-T-cell binding but also in sustaining DC-induced T-cell proliferation long after the initial contact has been established.

Effects of fatty acid-free bovine serum albumin and fetal calf serum supplementing repair cultures on pre- and post-warm viability of biopsied bovine embryos produced in vitro.

The objective of this study was to investigate the influence of fatty acid-free bovine serum albumin (BSA) or fetal calf serum (FCS) on the re-expansion of biopsied blastocysts and post-warm viability of subsequently vitrified embryos. Firstly, blastocysts produced in vitro were biopsied at Day 7 and cultured to allow repair in TCM199 with 0.3% BSA or 5% FCS for 24 h. The re-expansion rates and mean total numbers of cells of the re-expanded embryos after the repair culture with BSA were almost the same as that with FCS. Secondly, after biopsied embryos were similarly cultured for repair with BSA or FCS, re-expanded embryos were selected for vitrification. After warming and exposure to 0.5 M sucrose with 20% FCS in mPBS, the embryos were cultured in TCM199 with 5% FCS for 24 h. The re-expansion rate and mean total number of cells in re-expanded blastocysts in the BSA treatment group (97.4 +/- 2.9% and 106 +/- 42) was significantly higher than that in the FCS treatment group (51.6 +/- 9.1% and 61 +/- 38), respectively (P<0.05 and P<0.01). In conclusion, both FCS and BSA supplementation can be useful for repairing cultures of bovine biopsied blastocysts; but, compared with BSA supplementation, FCS supplementation during repair culture reduces the post-warm viability of biopsied and subsequently vitrified embryos.

Cyclin-dependent kinase inhibitor, p21Waf1, regulates vascular smooth muscle cell hypertrophy.

In the process of vascular diseases, smooth muscle cells (SMC) undergo not only hyperplasia but also hypertrophy, resulting in vascular remodeling. A cyclin-dependent kinase inhibitor (CDKI), p21Waf1, has been shown to play an important role in SMC hyperplasia. Here we investigated a potential role of p21Waf1 in SMC hypertrophy. An exposure of cultured rat SMC to serum drove the cell cycle progression with up-regulation of various cell cycle markers and increased activities of cyclin-dependent kinases, but did not cause SMC hypertrophy. In contrast, incubation of SMC for 48 h with angiotensin II (AII, 100 nmol/l) resulted in a significant increase in the cell size measured by flowcytometric forward-angle light scatter assay, in association with an increase in the ratio of [3H]leucine/[3H]thymidine uptake, indicating SMC hypertrophy. At 48 h, p21Waf1 expression was up-regulated in SMC exposed to AII but not in those exposed to serum. These results suggest that p21Waf1 may be involved in hypertrophy. To further investigate this issue, two manipulations of the p21Waf1 gene were performed. Adenovirus-mediated over-expression of p21Waf1 not only reduced S-phasic cells but also caused hypertrophy, despite the exposure to serum. Antisense oligodeoxynucleotide for p21Waf1 inhibited the hypertrophy of SMC exposed to AII. Our data suggest that p21Waf1 may play a role in SMC hypertrophy as well.

Polyamine synthesis inhibition attenuates vascular smooth muscle cell migration.

Vascular smooth muscle cell migration, occurring after intimal injury, is a substantial clinical problem in atherosclerosis and restenosis after stenting. Here we investigate the effects of polyamine synthesis inhibition on vascular smooth muscle cell migration after maximal and submaximal growth stimulation with PDGF-AB or FCS. Vascular smooth muscle cells were obtained from mouse aorta explants. These cells coexpressed smooth muscle alpha-actin, PDGFRalpha and PDGFRbeta as demonstrated by immunocytochemistry. Treatment with a high (100 ng/ml) concentration of PDGF-AB stimulated DNA synthesis 6-fold and markedly elevated cell migration. PDGF-AB (100 ng/ml) increased cellular spermidine concentration 2-fold, but had no effect on spermine or putrescine levels. Treatment with the polyamine synthesis inhibitors CGP48664 (1 microM) and DFMO (5 mM) prevented the PDGF-AB-induced increase in spermidine and reduced spermine concentrations, but had no effect on PDGF-AB-stimulated DNA synthesis or cell migration. Cell migration after submaximal stimulation with either PDGF-AB (8 ng/ml) or FCS (8%) was, however, inhibited by the polyamine synthesis blockers. In summary, these data show that polyamine synthesis inhibition attenuates vascular smooth muscle cell migration under submaximal growth-stimulating conditions, suggesting that polyamines participate in regulation of cell migration and that treatment with polyamine synthesis inhibitors might reduce vascular smooth muscle cell migration after intimal injury.

The alpha5beta1 integrin provides matrix survival signals for normal and osteoarthritic human articular chondrocytes in vitro.

OBJECTIVE: Chondrocyte cell death may play an important role in the development of arthritis. The goal of the present study was to evaluate the role of the extracellular matrix (ECM) in promoting chondrocyte survival via signals through the integrin family of ECM receptors. METHODS: Chondrocytes were isolated by sequential enzymatic digestion from normal ankle cartilage of organ donors and from osteoarthritic (OA) knee tissue obtained from patients undergoing total knee replacement. Cell survival in monolayer and in suspension culture was measured using fluorescent labels after treatment with specific integrin-blocking antibodies and echistatin, a disintegrin peptide. A quantitative enzyme-linked immunosorbent assay for histone-associated DNA fragments and morphologic evaluation by electron microscopy were used to evaluate apoptosis. RESULTS: Freshly isolated chondrocytes died when plated in serum-free media at low density on poly-L-lysine, but showed >95% survival on fibronectin (FN). A monoclonal blocking antibody to the alpha5-integrin subunit (FN receptor) significantly inhibited survival on FN, whereas control antibodies had no effect. Likewise, treatment of freshly isolated chondrocytes in serum-free alginate-suspension culture with the alpha5-blocking antibody resulted in cell death in a dose-dependent manner, with 20 microg/ml of the antibody reducing normal chondrocyte survival to 20% of that in controls, and OA chondrocyte survival to 23% of that in controls. Antibody inhibition of alphav and alpha1 integrins or treatment with echistatin did not cause cell death. Addition of insulin-like growth factor 1 (IGF-1; 100 ng/ ml) was not able to improve survival of alpha5-antibody-treated cells. However, treatment with 10% fetal bovine serum improved normal chondrocyte survival to 98% (a 5.1-fold increase) and OA chondrocyte survival to 64% (a 2.8-fold increase). Cell death due to alpha5 inhibition was associated with apoptosis. CONCLUSION: These results demonstrate that chondrocyte survival signals are transmitted via the alpha5beta1 FN receptor. Inhibition of matrix survival signals mediated by alpha5beta1 also inhibits the ability of IGF-1 to promote survival, suggesting that IGF-1-mediated survival signaling may require a cosignal from alpha5beta1.

Defined media for vitrification, warming, and rehydration: effects on post-thaw protein synthesis and viability of in vitro derived ovine embryos.

The purpose of this study was to assess the viability (rates of re-expanding and hatching in vitro), of in vitro derived ovine blastocysts using vitrification and warming/rehydration media containing fetal calf serum (20% FCS) or polyvinyl alcohol (0.1% PVA), and the incorporation of labelled methionine in protein synthesised during the first 4h after cryopreservation. In experiment 1, after 60 h culture in TCM-199 supplemented with 10% FCS, the hatching rates of blastocysts that had been vitrified, warmed, and rehydrated in media containing only PVA (p/p) were significantly (P<0.05) lower than those vitrified in medium containing PVA with warming and rehydration in medium containing FCS (p/s). Blastocysts that were vitrified in medium containing FCS and warmed and rehydrated in medium with PVA (s/p) had hatching rates that were significantly lower (P<0.01) than those vitrified, warmed, and rehydrated in media with only FCS (s/s). After warming, the number of dead cells in the p/p group was significantly (P<0.05) lower than in all other groups. In experiment 2, the [35S]methionine uptake by embryonic cells of the s/p group was significantly (P<0.01) higher than in other groups. The incorporation of labelled methionine into newly synthesised proteins was significantly lower in the p/p group (P<0.01) than in all other groups. No differences in the newly synthesised proteins were observed between groups. In conclusion, these results suggest that it is possible to replace serum with defined macromolecules in vitrification and warming/rehydration media for in vitro derived ovine blastocysts but this leads to a decrease in viability and a reduction in protein synthesis after warming.

Palmitate oxidation in rat hepatocytes is inhibited by foetal calf serum.

The rate of oxidation of fatty acids in mammals is minimal prior to birth. In this study, we have shown that foetal calf serum (FCS) inhibits oxidation of palmitate while serum from newborn calves is almost without effect. Foetal calf serum was also found to increase fatty acid synthesis from acetate. Uptake of laurate in mitochondria is partially dependent upon the carnitine palmitoyltransferase (CPT) I/CPT II system, while octanoate transport occurs without its participation. Comparison of the effects of FCS on the oxidation of palmitate, laurate and octanoate supports the view that the observed actions of FCS result from regulation of CPT I activity. The material in FCS that affects fatty acid metabolism has a molecular weight <3 kDa, as determined by dialysis and ultra-filtration studies.

FRAP-p70s6K signaling is required for pancreatic cancer cell proliferation.

BACKGROUND: FRAP-p70s6K signaling regulates mitogenic responses to growth factors in eukaryotic cells. Constitutive p70s6K activation occurs in some human malignancies and may contribute to dysregulated cell growth. We examined whether inhibition of this pathway affects mitogen-induced proliferation and cell cycle progression of human pancreatic cancer cells in vitro. METHODS: Quiescent BxPC3 and Panc-1 human pancreatic cancer cells treated with or without 20 ng/mL rapamycin (FRAP inhibitor) were repleted with 10% FCS to induce cell cycle entry. Proliferation was measured with MTT assay. Cell cycle and apoptosis were determined by FACS analysis. Phosphorylation of p70s6K, Akt, and cdc2 was evaluated by Western blot. Statistical analysis was by two-tailed t test (P < 0.05). RESULTS: Rapamycin (Rapa) inhibited the phosphorylation of p70s6K while inducing G(1) cell cycle arrest (P < 0.005). In both cell lines, Rapa inhibited serum-induced proliferation (P < 0.05) without affecting apoptosis. Cdc2 phosphorylation was inhibited by 15 min with Rapa (not shown), consistent with cell cycle arrest. Akt phosphorylation was not affected, indicating FRAP specificity of Rapa. CONCLUSIONS: FRAP-p70s6K signaling appears to be necessary for G(1)-to-S phase progression and proliferation in pancreatic cancer cells. This supports earlier work demonstrating a similar regulatory role for PI-3' kinase, an upstream activator of FRAP-p70s6K.

Polyamine regulation of ornithine decarboxylase and its antizyme in intestinal epithelial cells.

Ornithine decarboxylase (ODC) is feedback regulated by polyamines. ODC antizyme mediates this process by forming a complex with ODC and enhancing its degradation. It has been reported that polyamines induce ODC antizyme and inhibit ODC activity. Since exogenous polyamines can be converted to each other after they are taken up into cells, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone) (DEGBG), to block the synthesis of spermidine and spermine from putrescine and investigated the specific roles of individual polyamines in the regulation of ODC in intestinal epithelial crypt (IEC-6) cells. We found that putrescine, spermidine, and spermine inhibited ODC activity stimulated by serum to 85, 46, and 0% of control, respectively, in the presence of DEGBG. ODC activity increased in DEGBG-treated cells, despite high intracellular putrescine levels. Although exogenous spermidine and spermine reduced ODC activity of DEGBG-treated cells close to control levels, spermine was more effective than spermidine. Exogenous putrescine was much less effective in inducing antizyme than spermidine or spermine. High putrescine levels in DEGBG-treated cells did not induce ODC antizyme when intracellular spermidine and spermine levels were low. The decay of ODC activity and reduction of ODC protein levels were not accompanied by induction of antizyme in the presence of DEGBG. Our results indicate that spermine is the most, and putrescine the least, effective polyamine in regulating ODC activity, and upregulation of antizyme is not required for the degradation of ODC protein.

15-Deoxy-Delta(12,14)-prostaglandin J(2) facilitates thyroglobulin production by cultured human thyrocytes.

A cyclopentenone-type prostaglandin, 15-deoxy-Delta(12, 14)-prostaglandin J(2) (15-d-PGJ(2)), has been shown to induce the cellular stress response and to be a ligand for the peroxisome proliferator-activated receptor (PPAR)-gamma. We studied its effect on the basal and thyrotropin (TSH)-induced production of thyroglobulin (TG) by human thyrocytes cultured in the presence of 10% FBS. In 15-d-PGJ(2)-treated cells in which the agent itself did not stimulate cAMP production, both the basal production of TG and the response to TSH were facilitated, including the production of TG and cAMP, whereas such production was decreased in untreated cells according to duration of culture. PGD(2) and PGJ(2), which are precursors to 15-d-PGJ(2), exhibited an effect similar to 15-d-PGJ(2). However, the antidiabetic thiazolidinediones known to be specific ligands for PPAR-gamma, and WY-14643, a specific PPAR-alpha ligand, lacked this effect. 15-d-PGJ(2) and its precursors, but not the thiazolidinediones, induced gene expression for heme oxygenase-1 (HO-1), a stress-related protein, and strongly inhibited interleukin-1 (IL-1)-induced nitric oxide (NO) production. Cyclopentenone-type PGs have been recently shown to inhibit nuclear factor-kappaB (NF-kappaB) activation via a direct and PPAR-independent inhibition of inhibitor-kappaB kinase, suggesting that, in human thyrocytes, such PGs may inhibit IL-1-induced NO production, possibly via an inhibition of NF-kappaB activation. On the other hand, sodium arsenite, a known activator of the stress response pathway, induced HO-1 mRNA expression but lacked a promoting effect on TG production. Thus 15-d-PGJ(2) and its precursors appear to facilitate TG production via a PPAR-independent mechanism and through a different pathway from the cellular stress response that is available to cyclopentenone-type PGs. Our findings reveal a novel role of these PGs associated with thyrocyte differentiation.

Expression of receptor protein tyrosine phosphatase alpha mRNA in human prostate cancer cell lines.

Receptor protein tyrosine phosphatase alpha (RPTPalpha) is a transmembrane protein phosphatase, and has been proposed to be involved in the differentiation of the neuronal system. In the present study, we demonstrated the expression of RPTPalpha mRNA in several normal human tissues. We further investigated the regulation of expression of RPTPalpha mRNA in epithelial cells utilizing three commercially available human prostate cancer cell lines LNCaP, PC-3 and DU145. This is because these cells exhibit different levels of differentiation, defined by the expression of a tissue-specific differentiation antigen, prostatic acid phosphatase (PAcP), and their androgen sensitivity. LNCaP cells express PAcP and are androgen-sensitive cells, while PC-3 and DU145 cells do not express PAcP and are androgen-insensitive cells. Northern blot analyses revealed that, in LNCaP cells, fetal bovine serum (FBS) and 5alpha-dihydrotestosterone (DHT) down-regulates RPTPalpha mRNA expression, similar to the effect on PAcP. Contrarily, FBS up-regulated the RPTPalpha mRNA level in PC-3 and DU145 cells. In LNCaP cells, sodium butyrate inhibited cell growth and up-regulated RPTPalpha as well as PAcP mRNA expression. Although, sodium butyrate also inhibited the growth of PC-3 and DU145 cells, the level of RPTPalpha mRNA was decreased in PC-3, while increased in DU145 cells. Thus, data taken together indicate that the expression of RPTPalpha is apparently regulated by a similar mechanism to that of PAcP in LNCaP cells.

Polysorbate-80 coating enhances uptake of polybutylcyanoacrylate (PBCA)-nanoparticles by human and bovine primary brain capillary endothelial cells.

Certain drugs such as dalargin, loperamide or tubocurarine are not transported across the blood-brain barrier (BBB) and therefore exhibit no effects on the central nervous system. However, effects on the central nervous system can be observed when these drugs are loaded onto polybutylcyanoacrylate (PBCA)-nanoparticles and coated with polysorbate 80. The mechanism by which these complexed nanoparticles cross the BBB and exhibit their effects has not been elucidated. Cultured microvessel brain endothelial cells of human and bovine origin were used as an in vitro model for the BBB to gain further insight into the mechanism of uptake of nanoparticles. With cells from these species we were able to show that polysorbate 80-coated nanoparticles were taken up by brain endothelial cells much more rapidly and in significantly higher amounts (20-fold) than uncoated nanoparticles. The process of uptake was followed by fluorescence and confocal laser scanning microscopy. The results demonstrate that the nanoparticles are taken up by cells and that this uptake occurs via an endocytotic mechanism.

Purification and structure of neurostatin, an inhibitor of astrocyte division of mammalian brain.

Neurostatin was originally described as an inhibitor of astroblast and astrocytoma division present in rat brain extracts and immunologically related to the sugar moiety of epidermal growth factor receptor and to blood group antigens. It was purified recently from mammalian brain extracts and characterized as a glycosphingolipid, but its precise structure remained unknown. Neurostatin has now been purified to apparent homogeneity from ganglioside extracts of rat, bovine, and porcine brain. It is cytostatic for astroblasts, C6 glioma cells, and various human astrocytomas grades III and IV, with IC(50) values ranging from 250 to 450 nM, but does not affect the division of primary or transformed fibroblasts up to concentrations >4 microM. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of purified pig neurostatin showed a molecular ion of 1, 905 Da and ions of 1,863 and 1,934 Da, compatible with a disialoganglioside. Mono- and bidimensional NMR spectra, together with biochemical studies, suggest that neurostatin may be the 9-O-monoacetyl ester of GD1b.

Organoid culture of cannulated rat resistance arteries: effect of serum factors on vasoactivity and remodeling.

We developed an organoid culture technique to study the mechanisms involved in arterial remodeling. Resistance arteries were isolated from rat cremaster muscle and mounted in a pressure myograph at 75 mmHg. Vessels were studied during a 4-day culture period in DMEM with either 2% albumin, 10% heat-inactivated FCS (HI-FCS) or 10% dialyzed HI-FCS (12 kDa cut off) added to the perfusate. The albumin group showed a gradual loss of endothelial function and integrity, whereas smooth muscle agonist and myogenic responses were retained. No remodeling was observed. Vessels cultured in the presence of serum showed a progressive constriction. Smooth muscle responses and substance P-induced endothelium-dependent dilation were maintained. An inward remodeling of 17 +/- 4% in the HI-FCS group and 26 +/- 3% in the dialyzed HI-FCS group was found, while media cross-sectional areas were unchanged. These data show that pressurized resistance arteries can be maintained in culture for several days and undergo eutrophic remodeling in vitro in the presence of high molecular weight serum factors.

Induction of tissue plasminogen activator in differentiated NG108-15 cells.

Plasminogen activators may facilitate neurite outgrowth and neuronal migration in the developing nervous system. The expression of tissue plasminogen activator by NG108-15 neuroblastoma grown under a variety of conditions has been explored. High tissue plasminogen mRNA expression correlates with growth conditions which induce morphological differentiation and neurite outgrowth; however, NG108-15 cells grown in suspension with dibutyryl-cAMP also show a high level of tissue plasminogen activator expression.

Estradiol inhibits smooth muscle cell growth in part by activating the cAMP-adenosine pathway.

Estradiol inhibits smooth muscle cell growth; however, the mechanisms involved remain unclear. Because estradiol stimulates cAMP synthesis and adenosine inhibits cell growth, we hypothesized that the conversion of cAMP to adenosine (ie, the cAMP-adenosine pathway) mediates in part the inhibitory effects of estradiol on vascular smooth muscle cell growth. To test this hypothesis, we examined the effects of estradiol (0.001 to 1 micromol/L) on serum-induced DNA, collagen, and total protein synthesis and cell number in the absence and presence of 1, 3-dipropyl-8-p-sulfophenylxanthine (10 nmol/L; A(1)/A(2) adenosine receptor antagonist), KF17837 (10 nmol/L; selective A(2) adenosine receptor antagonist), 8-cyclopentyl-1,3-dipropylxanthine (10 nmol/L; selective A(1) adenosine receptor antagonist), and 2', 5'-dideoxyadenosine (10 micromol/L; adenylyl cyclase inhibitor). Estradiol inhibited all measures of cell growth, and the concentration-dependent inhibitory curves for estradiol were shifted to the right (P<0.05) by 1,3-dipropyl-8-p-sulfophenylxanthine, KF17837, and 2',5'-dideoxyadenosine but not by 8-cyclopentyl-1, 3-dipropylxanthine. Moreover, the inhibitory effects of estradiol were enhanced by stimulation of adenylyl cyclase with forskolin and by inhibition of adenosine metabolism with erythro-9-(2-hydroxy-3-nonyl)adenine plus iodotubericidin (adenosine deaminase and kinase inhibitors, respectively). Estradiol also increased levels of cAMP and adenosine, and these effects were blocked by 2',5'-dideoxyadenosine (P<0.05). Our results support the hypothesis that estradiol stimulates cAMP synthesis and cAMP-derived adenosine regulates smooth muscle cell growth via A(2) adenosine receptors. Thus, the cAMP-adenosine pathway may contribute importantly to the antivasooclusive effects of estradiol.