Association between the activity of the system A amino acid transporter in the microvillous plasma membrane of the human placenta and severity of fetal compromise in intrauterine growth restriction.

Primarily, our objectives were to compare system A amino acid transporter activity in the microvillous plasma membrane (MVM) of placentas from normally grown (appropriate for gestational age, AGA) and intrauterine growth-restricted (IUGR) fetuses delivered during the third trimester, as a whole and in relation to the severity of IUGR. Ten AGA and 16 IUGR pregnancies were studied at the time of elective cesarean section performed between 28 and 40 wk of gestation. Severity of IUGR pregnancies was assessed primarily by Doppler velocimetry and fetal heart rate monitoring. Placental MVM vesicles were prepared, and system A activity in these was measured. The transporter activity was significantly lower in IUGR compared with AGA pregnancies. Within the IUGR group system A activity was only significantly lower, compared with AGA, in cases that presented with a reduction in umbilical blood flow. We conclude that placental MVM system A activity is lower in IUGR compared with AGA pregnancies delivered during the third trimester. System A activity is related to the severity of IUGR.

Substratum-dependent stimulation of fibroblast migration by the gelatin-binding domain of fibronectin.

Nanomolar concentrations of native fibronectin and its RGDS-containing cell-binding domain have previously been reported to stimulate fibroblast migration in the transmembrane (or 'Boyden chamber') assay; in contrast, the gelatin-binding domain (GBD) of fibronectin has consistently been reported to be devoid of migration-stimulating activity in this assay. We have examined the effects of fibronectin and several of its purified functional domains on the migration of human skin fibroblasts in what is presumably a more physiologically relevant assay involving the movement of cells into a 3-D matrix of native type I collagen fibrils. We report that: (a) femtomolar concentrations of GBD stimulate fibroblast migration into such collagen matrices; and (b) fibronectin, as well as peptides containing all other of its functional domains, do not exhibit migration-stimulating activity when tested in the femtomolar to nanomolar concentration range (i.e. 0.1 pg/ml to 1 microgram/ml). The correct assignment of migration-stimulating activity to GBD, rather than to a contaminant, was confirmed by: (a) the use of several fibronectin and GBD purification protocols; (b) the neutralization of GBD migration-stimulating activity by monoclonal antibodies directed against epitopes present in this domain; (c) the time-dependent generation of migration-stimulating activity by the proteolytic degradation of native fibronectin; and (d) obtaining an identical dose-response curve with a genetically engineered GBD peptide. The cryptic migration-stimulating activity of GBD was not affected by the presence of serum or native fibronectin, but was inhibited by TGF-beta 1. Parallel experiments using the transmembrane assay confirmed that GBD was devoid of migration-stimulating activity in this assay when membranes coated with gelatin were used, but revealed that significant stimulation of migration was achieved with membranes coated with native type I collagen. Cells preincubated with GBD for 24 hours whilst growing on plastic tissue culture dishes and then plated onto native collagen matrices in the absence of further GBD also displayed an elevated migration compared to controls. Taken together, these observations suggest that: (a) the interaction of GBD with a putative cell surface receptor (and not the collagen substratum) initiates a persistent alteration in cell phenotype which is manifest by an increase in migratory activity when these cells are cultured on a native collagen substratum; and (b) GBD may play a hitherto unrecognised role in the control of cell migration in response to the local release of proteases during pathological processes, such as tumour invasion and wound repair.

Embryonal rhabdomyosarcoma of the orbit in a 35-year-old man.

A 35-year-old man had rapidly progressive proptosis of the right eye with associated chemosis over a period of several weeks. Computed tomography demonstrated a solid extraconal mass in the inferior anterior right orbit. Pathologic examination revealed the lesion to be an embryonal rhabdomyosarcoma. Consistent with the diagnosis, immunohistochemical assays demonstrated positive staining with myoglobin, desmin, and muscle-specific actin. The lesion grew rapidly and was further surgically excised. Subsequently, treatment with radiation and chemotherapy was initiated. Primary embryonal rhabdomyosarcoma of the orbit is an extremely rare tumor in adults, and, to our knowledge, this patient represents the oldest individual reported to have developed such a tumor, as documented by immunohistochemical analysis.

Migration stimulating factor (MSF): its structure, mode of action and possible function in health and disease.

We have previously reported that (a) fetal fibroblasts migrate into 3-dimensional collagen matrices to a significantly greater extent that do adult cells, (b) this difference in migratory behaviour results from the secretion by fetal fibroblasts of a "migration stimulating factor" (MSF), and (c) adult fibroblasts retain responsiveness to MSF, this providing the basis of a bioassay for monitoring factor activity. Using a recently modified purification protocol, MSF isolated from fetal fibroblast conditioned medium elutes as a single activity peak in the penultimate Mono Q anion exchange chromatography step. Analysis of this material by SDS-PAGE indicates that it consists of three proteins, one with an apparent molecular mass of 119 kDa and a doublet with molecular masses of approximately 43 and 33 kDa, respectively. Our data suggest that the two proteins comprising the doublet result from the degradation of the larger molecule during the purification procedure. Both the 119 kDa species and lower molecular weight doublet stimulate fibroblast migration (with half maximal activity in the region of 1-10 pg/ml) and contain a structural domain exhibiting significant amino acid sequence homology with the gelatin-binding fragment (GBF) of fibronectin. Bona fide preparations of GBF, obtained by the limited proteolysis of plasma fibronectin, also stimulate the migration of adult fibroblasts in a similar dose-dependent manner to that of MSF. In spite of this similarity, MSF and GBF differ in terms of a number of biological and biochemical parameters, thereby suggesting that MSF is a distinct gene product and not a proteolytic degradation fragment of fibronectin. MSF stimulates the synthesis of a high molecular weight species of hyaluronic acid (HA). Our current data suggest that the observed effect of MSF on cell migration is actually a secondary consequence of the accumulation of this HA in the collagen matrix. TGF-beta is a potent inhibitor of MSF, both in terms of its effects on cell migration and HA synthesis. As MSF is present in wound fluid, we have suggested that the inhibition of MSF activity by TGF-beta may reflect the antagonistic interaction of these two cytokines in the control of the wound healing process. Our recent data indicate that discrete minority subpopulations of MSF-secreting fibroblasts are also present at specific sites in the healthy adult and that these may undergo a transient and local expansion during wound healing.(ABSTRACT TRUNCATED AT 400 WORDS)

Detection of migration stimulating activity in wound fluid.

Fetal skin fibroblasts produce a soluble "migration stimulating factor" (MSF) which is not made by their normal adult counterparts. MSF stimulates the migration of adult skin fibroblasts into 3D collagen gels, thus providing the basis of a convenient bioassay for its presence. We have previously reported that MSF stimulates hyaluronic acid (HA) synthesis by adult skin fibroblasts and that this effect on matrix deposition appears to be responsible for the observed increase in cell motility. In the present study, wound fluid samples were collected from 18 patients undergoing surgery for various nonmalignant conditions and these were then fractionated according to the protocol used to isolate MSF from fetal fibroblast-conditioned medium. Detectable levels of migration stimulating activity were present in 17/18 (94%) of these samples. Paired serum samples obtained both pre- and postoperatively from five patients with positive wound fluid samples were also analyzed for MSF activity; such activity was found in only 1/5 (20%) of the preoperative and 0/5 (0%) of the postoperative serum samples. These data suggest that the MSF present in wound fluid is not derived from a plasma transudate or from platelet degranulation, but may reflect the transient and localized reinitiation of MSF production by adult fibroblasts in response to wounding. Taken together with previous observations regarding the effect of MSF on fibroblast migration and HA synthesis, our data suggest a possible physiological function of MSF in the wound healing response. Previous studies have revealed that MSF is produced by a subpopulation of apparently persistent fetal-like skin fibroblasts obtained from breast cancer patients and is also found in the serum of these individuals. Wound fluid and serum samples were accordingly collected from patients undergoing surgery for various types of malignant conditions or with a previous history of cancer; detectable levels of MSF activity were found in 8/10 (80%) of these wound fluid samples, 2/3 (66.6%) of the preoperative, and 3/3 (100%) of the postoperative paired serum samples. These findings suggest that the presence of detectable serum levels of MSF is not restricted to breast cancer and may be a general feature of malignant disease.

Antagonistic effects of TGF-beta 1 and MSF on fibroblast migration and hyaluronic acid synthesis. Possible implications for dermal wound healing.

The migration of adult skin fibroblasts into three-dimensional collagen gel matrices is differentially affected by cell density, with subconfluent cells displaying a significantly elevated level of migration compared to confluent ones. Fetal fibroblasts differ from adult cells in that they display an elevated level of migration at both subconfluent and confluent cell densities. We have previously reported that this difference in behaviour results from the secretion by fetal fibroblasts of a 'migration stimulating factor' (MSF) which is not made by their normal adult counterparts, and that MSF appears to act by stimulating the synthesis of hyaluronic acid (HA). Data presented in this communication indicate that (a) MSF specifically stimulates the synthesis of high molecular weight species of HA, (b) TGF-beta 1 inhibits the elevated migration of adult fibroblasts plated at subconfluent cell density, (c) under these conditions, TGF-beta 1 induces a parallel decrease in the synthesis of high molecular weight HA and increase in the synthesis of low molecular weight HA, (d) TGF-beta 1 is a potent antagonist of MSF, effectively blocking its stimulation of cell migration and synthesis of high molecular weight HA, and (e) the inhibition of fibroblast migration by TGF-beta 1 does not appear to be a chemotactic response dependent upon the existence of a concentration gradient of the cytokine. Our observations regarding the inhibitory effects of TGF-beta 1 on fibroblast migration into 3D collagen gels stand in marked contrast to various published reports indicating that this cytokine stimulates the migration of human skin fibroblasts through the pores of polycarbonate filters as used in modified Boyden chamber assays; this discrepancy underscores the importance of the substratum in modulating cellular response to cytokines. Our results are discussed in terms of the possible combined contribution of MSF and TGF-beta 1 to wound healing.

Heterogeneity amongst fibroblasts in the production of migration stimulating factor (MSF): implications for cancer pathogenesis.

Fetal skin fibroblasts migrate into 3D collagen gels to a significantly greater extent than do adult cells. This enhanced motility of fetal fibroblasts appears to result from the production of a "migration stimulating factor" (MSF) which is not made by their normal adult counterparts. Adult skin fibroblasts retain responsiveness to MSF and cells exposed to this factor achieve the elevated levels of migration characteristic of fetal cells. MSF has been purified to homogeneity, has an apparent molecular mass of 70 kD and has been further characterized in terms of a number of biochemical parameters. Studies concerned with the mechanism of action of MSF indicate that it stimulates the production of a high molecular weight class of hyaluronic acid (HA). Concurrent exposure of cells to Streptomyces hyaluronidase blocks the stimulation of adult fibroblast migration by MSF. In a related series of experiments, we have shown that TGF-beta inhibits the effects of MSF on both cell migration and HA production. Taken together, these data suggest that the stimulation of fibroblast migration by MSF is dependent upon (and may directly result from) a primary induction of HA synthesis. We have previously reported that skin fibroblasts obtained from patients with sporadic and familial breast cancer, as well as the unaffected first-degree relatives of familial breast cancer patients, commonly display a fetal-like migratory phenotype. Subsequent work has indicated that (a) these fetal-like cells also produce MSF, and (b) detectable levels of MSF are present in the serum of sporadic breast cancer patients both prior to and following surgical resection of the primary tumor mass. On the basis of these and related observations, we have put forward an hypothesis suggesting that the disruption in normal epithelial-mesenchymal interactions caused by the persistent production of MSF by fibroblasts in the adult may contribute directly to the pathogenesis of an epithelial cancer. The demonstration of aberrant fibroblasts in sporadic cancer patients (both in our own and independent studies) is not consistent with the "germ-line genetic lesion" model commonly invoked to account for the presence of such cells in patients with hereditary cancer syndromes.(ABSTRACT TRUNCATED AT 400 WORDS)

Migration stimulating activity in serum of breast cancer patients.

An assay to measure the ability to stimulate migration of fibroblasts into collagen gel was carried out on serum from treated and untreated breast cancer patients and from healthy controls. Migration stimulating activity was found in the serum of 10 (83%) of 12 untreated breast cancer patients immediately before surgical resection of the primary tumour and in 9 (75%) of them 4 days after resection; in 13 (93%) of 14 patients 1-13 years after tumour resection who had received adjuvant treatment; and in 2 (10%) of 20 healthy women matched for age. The migration stimulating activity in cancer patients' serum was indistinguishable from the migration stimulating factor produced in vitro by fetal and cancer patient skin fibroblasts in its behaviour in various biochemical fractionation procedures. The presence of this activity in the serum of treated breast cancer patients clearly distinguishes it from other oncofetal proteins, which all seem to be produced by tumours.

Mechanism of action of the migration stimulating factor produced by fetal and cancer patient fibroblasts: effect on hyaluronic and synthesis.

We have previously demonstrated that confluent fetal fibroblasts migrate into three-dimensional collagen gels to a significantly greater extent than their normal adult counterparts. Recent studies have revealed that this behavioral difference results from the secretion by fetal fibroblasts of a soluble migration-stimulating factor (MSF) which acts on these cells in an autocrine fashion. Adult fibroblasts do not produce MSF but remain responsive to it. Skin fibroblasts from cancer patients resemble fetal fibroblasts (rather than normal adult cells) with respect to their migratory behavior on collagen gels and continued production of MSF. This communication is concerned with elucidating the biochemical basis of MSF activity. Data are presented indicating that a) hyaluronic acid is required for the elevated migratory activity displayed by confluent fetal and breast cancer patient skin fibroblast; b) adult fibroblasts exhibit a bell-shaped dose-response to MSF, with maximal stimulation of migration observed at a concentration of 10 ng/ml; c) the migratory activity of adult fibroblasts pre-incubated with MSF remains high in the absence of additional factor: and d) MSF affects both the quantity and size class distribution of hyaluronic acid synthesized by adult fibroblasts. We have previously speculated that the persistent fetal-like fibroblasts of breast cancer patients play a direct role in disease pathogenesis by perturbing normal epithelial-mesenchymal interactions. The observations reported here suggest that MSF-induced alterations in hyaluronic acid synthesis may contribute to the molecular basis of such perturbations.

Purification of the migration stimulating factor produced by fetal and breast cancer patient fibroblasts.

We have previously shown that (i) human skin fibroblasts of fetal and adult origin display distinctive migratory phenotypes, (ii) this difference in cell behavior results from the production of a soluble "migration stimulating factor" (MSF) by fetal cells, and (iii) skin fibroblasts from breast cancer patients commonly resemble fetal fibroblasts both in migratory phenotype and in production of MSF. Data are now presented indicating that MSF present in the conditioned medium of fetal and cancer patient fibroblasts is precipitated at 10% saturation ammonium sulfate and binds to heparin and cation-exchange resins. Based on this information, we have devised a scheme for the purification of MSF involving the sequential application of ammonium sulfate precipitation, heparin affinity, gel filtration, and reverse-phase chromatography. Purified MSF has an estimated molecular mass of 70 kDa; amino acid analysis reveals a relatively high level of proline (13.34 residues per 100). Our results further suggest that skin fibroblasts from breast cancer patients produce an additional factor with migration stimulating activity; this factor is precipitated at higher concentrations of ammonium sulfate and binds to anion-exchange resins. We have previously discussed the possible direct involvement of fetal-like fibroblasts in cancer pathogenesis. The availability of MSF obtained from cancer patient fibroblasts provides a potential means with which to examine the complex cellular interactions contributing to this process as well as develop a screening regime for identifying individuals at elevated risk of developing cancer.

Foetal and cancer patient fibroblasts produce an autocrine migration-stimulating factor not made by normal adult cells.

We have previously reported that (1) the migration of foetal and adult fibroblasts into three-dimensional collagen matrices is differentially affected by cell density, and (2) skin fibroblasts from cancer patients commonly display a foetal-like mode of migratory behaviour. Data presented here indicate that differences in the migration of these cell types are particularly apparent in cultures plated at high density (i.e. at cell confluence); under these conditions, foetal fibroblasts and the foetal-like fibroblasts of cancer patients migrate into the three-dimensional collagen matrix to a significantly greater extent than do normal adult cells. In this initial study concerned with the biochemical basis of these observations, we report that medium conditioned by either foetal or cancer patient fibroblasts stimulates the migration of confluent adult cells. This stimulation of migration is specific to confluent cells, as the migration of subconfluent adult fibroblasts is unaffected by these conditioned media. Gel filtration chromatography of foetal fibroblast-conditioned medium indicates that migration-stimulating activity is recovered in a single peak with an apparent molecular mass in the range of 50-60 (X 10(3]. The active migration stimulating factor (MSF) in both foetal and cancer patient fibroblast-conditioned media appears to be a protein stable at acid pH, but inactivated by heat, alkaline pH and reductive alkylation. MSF produced by foetal and cancer patient fibroblasts is presumably responsible for the characteristically elevated levels of migration displayed by these cells in confluent culture, thereby suggesting an autocrine mode of action for this factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidic phospholipid species inhibit adenylate cyclase activity in rat liver plasma membranes.

Incubation of rat liver plasma membranes with liposomes of dioleoyl phosphatidic acid (dioleoyl-PA) led to an inhibition of adenylate cyclase activity which was more pronounced when fluoride-stimulated activity was followed than when glucagon-stimulated activity was followed. If Mn2+ (5 mM) replaced low (5 mM) [Mg2+] in adenylate cyclase assays, or if high (20 mM) [Mg2+] were employed, then the perceived inhibitory effect of phosphatidic acid was markedly reduced when the fluoride-stimulated activity was followed but was enhanced for the glucagon-stimulated activity. The inhibition of adenylate cyclase activity observed correlated with the association of dioleoyl-PA with the plasma membranes. Adenylate cyclase activity in dioleoyl-PA-treated membranes, however, responded differently to changes in [Mg2+] than did the enzyme in native liver plasma membranes. Benzyl alcohol, which increases membrane fluidity, had similar stimulatory effects on the fluoride- and glucagon-stimulated adenylate cyclase activities in both native and dioleoyl-PA-treated membranes. Incubation of the plasma membranes with phosphatidylserine also led to similar inhibitory effects on adenylate cyclase and responses to Mg2+. Arrhenius plots of both glucagon- and fluoride-stimulated adenylate cyclase activity were different in dioleoyl-PA-treated plasma membranes, compared with native membranes, with a new 'break' occurring at around 16 degrees C, indicating that dioleoyl-PA had become incorporated into the bilayer. E.s.r. analysis of dioleoyl-PA-treated plasma membranes with a nitroxide-labelled fatty acid spin probe identified a new lipid phase separation occurring at around 16 degrees C with also a lipid phase separation occurring at around 28 degrees C as in native liver plasma membranes. It is suggested that acidic phospholipids inhibit adenylate cyclase by virtue of a direct headgroup specific interaction and that this perturbation may be centred at the level of regulation of this enzyme by the stimulatory guanine nucleotide regulatory protein NS.

The action of islet activating protein (pertussis toxin) on insulin's ability to inhibit adenylate cyclase and activate cyclic AMP phosphodiesterases in hepatocytes.

Treatment of hepatocytes with islet activating protein (pertussis toxin) from Bordetella pertussis blocked the ability of insulin to inhibit adenylate cyclase activity both in broken plasma membranes and in intact hepatocytes. Such treatment of intact hepatocytes with pertussis toxin did not prevent insulin from activating the peripheral plasma membrane cyclic AMP phosphodiesterase although it did inhibit the ability of insulin to activate the 'dense-vesicle' cyclic AMP phosphodiesterase. The ability of glucagon pretreatment of hepatocytes to block insulin's activation of the plasma membrane cyclic AMP phosphodiesterase was abolished in pertussis toxin-treated hepatocytes. It is suggested that the ability of insulin to manipulate cyclic AMP concentrations by inhibiting adenylate cyclase and activating the plasma membrane and 'dense-vesicle' cyclic AMP phosphodiesterases involves interactions with the guanine nucleotide regulatory protein system occurring in liver plasma membranes.