The <em>growth</em> autonomy of human tumor cells is considered due to the endogenous production of <em>growth</em> <em>factors</em>. Transcriptional expression of candidates for autocrine stimulatory <em>factors</em> such as basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF), acidic FGF, and transforming <em>growth</em> <em>factor</em> type beta were determined in human brain tumors. Basic FGF was expressed abundantly in 17 of 18 gliomas, <em>20</em> of 22 meninglomas, and 0 of 5 metastatic brain tumors. The level of mRNA expression of acidic FGF in gliomas was significant. In contrast, transforming <em>growth</em> <em>factor</em> type beta 1 was expressed in all the samples investigated. The mRNA for basic FGF and its peptide were localized in tumor cells in vivo by in situ hybridization and immunohistochemistry, showing that basic FGF is actually produced in tumor cells. Our results suggest that tumor-derived basic FGF is involved in the progression of gliomas and meningiomas in vivo, whereas acidic FGF is expressed in a tumor origin-specific manner, suggesting that acidic FGF works in tandem with basic FGF in glioma tumorigenesis.

The role of immune cell products in modulating connective tissue metabolism was investigated. Supernates of both unstimulated and phytohemagglutinin-stimulated human mononuclear cell cultures suppressed <em>fibroblast</em> proliferation (up to 90%) and concomintantly stimulated <em>fibroblast</em> prostaglandin E(PGE) synthesis (<em>20</em>- to 70-fold). The <em>growth</em> suppression was, at least in part, a secondary result of the increased <em>fibroblast</em> PGE synthesis; <em>growth</em> suppression (a) paralled the increased <em>fibroblast</em> PGE synthesis, (b) was reversed by addition of inhibitors of prostaglandin synthesis (indomethacin, meclofenamate, and eicostaetraynoic acid), and (c) was reproduced by addition of exogenous PGE2 to <em>fibroblast</em> cultures. The prostaglandin-stimulatory, <em>growth</em>-suppressive activity was a product of non-T-lymphocyte, adherent cells and was present within 6 h of mononuclear cell culture. The activity was heat (56 degrees C) and trypsin sensitive, nondialyzable, and appeared in the 12,000-<em>20</em>,000 mol wt fractions by Sephadex G-100 chromatography. The activity in supernates of mononuclear cell cultures was removed by incubation with <em>fibroblasts</em> but not by similar incubation with peripheral blood mononuclear cells. Mononuclear cells release a <em>factor</em>(s) which modulates <em>fibroblast</em> proliferation by altering prostaglandin metabolism.

The aim of this study was to identify fibrogenic mediators stimulating activation, proliferation, and/or matrix synthesis of rat pancreatic stellate cells (PSC). PSC were isolated from the pancreas of normal Wistar rats and from rats with cerulein pancreatitis. Cell activation was demonstrated by immunofluorescence microscopy of smooth muscle alpha-actin (SMA) and real-time quantitative RT-PCR of SMA, fibronectin, and transforming <em>growth</em> <em>factor</em> (TGF)-beta(1). Proliferation was measured by bromodeoxyuridine incorporation. Matrix synthesis was demonstrated on the protein and mRNA level. Within a few days in primary culture, PSC changed their phenotype from fat-storing to SMA-positive myo<em>fibroblast</em>-like cells expressing platelet-derived <em>growth</em> <em>factor</em> (PDGF) alpha- and PDGF beta-receptors. TGF-beta(1) and tumor necrosis <em>factor</em> (TNF)-alpha accelerated the change in the cells' phenotype. Addition of 50 ng/ml PDGF and 5 ng/ml basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) to cultured PSC significantly stimulated cell proliferation (4.37 +/- 0.49- and 2.96 +/- 0.39-fold of control). Fibronectin synthesis calculated on the basis of DNA was stimulated by 5 ng/ml bFGF (3.44 +/- 1.13-fold), 5 ng/ml TGF-beta(1) (2.46 +/- 0.89-fold), <em>20</em> ng/ml PDGF (2.27 +/- 0.68-fold), and 50 ng/ml TGF-alpha (1.87 +/- 0.19-fold). As shown by RT-PCR, PSC express predominantly the splice variant EIII-A of fibronectin. Immunofluorescence microscopy and Northern blot confirmed that in particular bFGF and TGF-beta(1) stimulated the synthesis of fibronectin and collagens type I and III. In conclusion, our data demonstrate that 1) TGF-beta(1) and TNF-alpha accelerate the change in the cell phenotype, 2) PDGF represents the most effective mitogen, and 3) bFGF, TGF-beta(1), PDGF, and, to a lesser extent, TGF-alpha stimulate extracellular matrix synthesis of cultured rat PSC.

OBJECTIVE

Disturbances in mineral metabolism define an increased cardiovascular risk in patients with chronic kidney disease. Fibroblast growth factor-23 (FGF23) is a circulating regulator of phosphate and vitamin D metabolism and has recently been implicated as a putative pathogenic factor in cardiovascular disease. Because other members of the FGF family play a role in lipid and glucose metabolism, we hypothesized that FGF23 would associate with metabolic factors that predispose to an increased cardiovascular risk. The goal of this study was to investigate the relationship between FGF23 and metabolic cardiovascular risk factors in the community.

RESULTS

Relationships between serum FGF23 and body mass index (BMI), waist circumference, waist-to-hip ratio, serum lipids, and fat mass were examined in 2 community-based, cross-sectional cohorts of elderly whites (Osteoporotic Fractures in Men Study: 964 men aged 75±3.2; Prospective Investigation of the Vasculature in Uppsala Seniors study: 946 men and women aged 70). In both cohorts, FGF23 associated negatively with high-density lipoprotein and apolipoprotein A1 (7% to 21% decrease per 1-SD increase in log FGF23; P<0.01) and positively with triglycerides (11% to 14% per 1-SD increase in log FGF23; P<0.01). A 1-SD increase in log FGF23 was associated with a 7% to 20% increase in BMI, waist circumference, and waist-to-hip ratio and a 7% to 18% increase in trunk and total body fat mass (P<0.01) as determined by whole-body dual x-ray absorptiometry. FGF23 levels were higher in subjects with the metabolic syndrome compared with those without (46.4 versus 41.2 pg/mL; P<0.05) and associated with an increased risk of having the metabolic syndrome (OR per 1-SD increase in log FGF23, 1.21; 95% CI, 1.04 to 1.40; P<0.05).

CONCLUSIONS

We report for the first time on associations between circulating FGF23, fat mass, and adverse lipid metabolism resembling the metabolic syndrome, potentially representing a novel pathway(s) linking high FGF23 to an increased cardiovascular risk.

Platelet-derived <em>growth</em> <em>factor</em> (PDGF) has previously been shown to bind to a specific high affinity receptor on human foreskin <em>fibroblasts</em>. The present study was carried out to characterize some of the cellular events resulting from the interaction of the ligand with its receptor. Radiolabeled PDGF was rapidly internalized and degraded after binding to the cells. The degradation was complete and was inhibited by low concentrations of the lysosomotropic agents, chloroquine, ammonium chloride, or methylamine, suggesting that the degradation occurs in the lysosomes. The cellular binding capacity for PDGF decreased after exposure of the cells to PDGF at 37 degrees C. This down regulation of the PDGF receptor was optimal after a 60-min incubation at 37 degrees C and half-maximal at 0.5 nM concentration of PDGF. The binding capacity was restored when the PDGF-containing medium was changed to medium without PDGF; the binding capacity increased from 40 to 80% od the initial value after a 4-h incubation at 37 degrees C. The reappearance on the cell surface of PDGF-binding sites was dependent on protein synthesis and totally blocked by cycloheximide (<em>20</em> micrograms/ml). Thus, either the receptor has to be resynthesized after internalization or, alternatively, any step in the recycling of "used" receptors is dependent on protein synthesis. Exposure of the cells to PDGF also caused a dose-dependent decrease in the binding capacity for epidermal <em>growth</em> <em>factor</em> which has a distinct receptor on these cells. In contrast, epidermal <em>growth</em> <em>factor</em> did not modulate the PDGF binding capacity, lending no support to the idea that the receptors for epidermal <em>growth</em> <em>factor</em> and PDGF are processed in a common pathway.

BACKGROUND

Fibroblast growth factor (FGF) 21, a novel member of the FGF family, plays a role in a variety of endocrine functions, including regulation of glucose and lipid metabolism. The role of FGF21 in skeletal muscle is currently not known.

METHODS

Serum levels and skeletal muscle mRNA of FGF21 were determined in normal glucose tolerant (n = 40) and type 2 diabetic (T2D; n = 40) subjects. We determined whether FGF21 has direct effects on glucose metabolism in cultured myotubes (n = 8) and extensor digitorum longus skeletal muscle.

RESULTS

Serum FGF21 levels increased 20% in T2D versus normal glucose tolerant subjects (p < 0.05), whereas skeletal muscle mRNA expression was unaltered. Fasting insulin, homeostatic model assessment of insulin resistance (HOMA-IR), waist circumference, and body mass index (BMI) significantly correlated with serum FGF21 levels in T2D (p < 0.01), but not in normal glucose tolerant subjects. Serum FGF21 concentrations were greater in T2D patients in the highest tertile of fasting insulin (p < 0.05) and BMI (p < 0.05). Stepwise regression analysis identified BMI as the strongest independent variable correlating with FGF21. FGF21 exposure increased basal and insulin-stimulated glucose uptake in human myotubes, coincident with increased glucose transporter 1 mRNA, and enhanced glucose transporter 1 abundance at the plasma membrane. In isolated extensor digitorum longus muscle, FGF21 potentiated insulin-stimulated glucose transport, without altering phosphorylation of Akt or AMP-activated protein kinase.

CONCLUSIONS

Plasma FGF21 is increased in T2D patients, and positively correlated with fasting insulin and BMI. However, FGF21 has direct effects in enhancing skeletal muscle glucose uptake, providing additional points of regulation that may contribute to the beneficial effects of FGF21 on glucose homeostasis. Whether increased plasma FGF21 in T2D is a compensatory mechanism to increase glucose metabolism remains to be determined.

The family with sequence similarity <em>20</em>, member C (Fam<em>20</em>C) has recently been identified as the Golgi casein kinase. Fam<em>20</em>C phosphorylates secreted proteins on Ser-x-Glu/pSer motifs and loss-of-function mutations in the kinase cause Raine syndrome, an often-fatal osteosclerotic bone dysplasia. Fam<em>20</em>C is potentially an upstream regulator of the phosphate-regulating hormone <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23), because humans with FAM<em>20</em>C mutations and Fam<em>20</em>C KO mice develop hypophosphatemia due to an increase in full-length, biologically active FGF23. However, the mechanism by which Fam<em>20</em>C regulates FGF23 is unknown. Here we show that Fam<em>20</em>C directly phosphorylates FGF23 on Ser(180), within the FGF23 R(176)XXR(179)/S(180)AE subtilisin-like proprotein convertase motif. This phosphorylation event inhibits O-glycosylation of FGF23 by polypeptide N-acetylgalactosaminyltransferase 3 (GalNAc-T3), and promotes FGF23 cleavage and inactivation by the subtilisin-like proprotein convertase furin. Collectively, our results provide a molecular mechanism by which FGF23 is dynamically regulated by phosphorylation, glycosylation, and proteolysis. Furthermore, our findings suggest that cross-talk between phosphorylation and O-glycosylation of proteins in the secretory pathway may be an important mechanism by which secreted proteins are regulated.

Abnormalities of kidney and urinary tract development are the most common cause of end-stage kidney failure in childhood in the United States. Over the past <em>20</em> years, the advent of mutant and transgenic mice and the manipulation of gene expression in other animal models has resulted in major advances in identification of the cellular and molecular mechanisms that direct kidney morphogenesis, providing insights into the pathophysiology of renal and urologic anomalies. This review focuses on the molecular mechanisms that define kidney progenitor cell populations, induce nephron formation within the metanephric mesenchyme, initiate and organize ureteric bud branching, and participate in terminal differentiation of the nephron. Highlighted are common signaling pathways that function at multiple stages during kidney development, including signaling via Wnts, bone morphogenic proteins, <em>fibroblast</em> <em>growth</em> <em>factor</em>, sonic hedgehog, RET/glial cell-derived neurotrophic <em>factor</em>, and notch pathways. Also emphasized are the roles of transcription <em>factors</em> Odd1, Eya1, Pax2, Lim1, and WT-1 in directing renal development. Areas requiring future investigation include the <em>factors</em> that modulate signaling pathways to provide temporal and site-specific effects. The evolution of our understanding of the cellular and molecular mechanisms of kidney development may provide methods for improved diagnosis of renal anomalies and, hopefully, targets for intervention for this common cause of childhood end-stage kidney disease.

Influenza A viruses continue to cause widespread morbidity and mortality. There is an added concern that the highly pathogenic H5N1 influenza A viruses, currently found throughout many parts of the world, represent a serious public health threat and may result in a pandemic. Intervention strategies to halt an influenza epidemic or pandemic are a high priority, with an emphasis on vaccines and antiviral drugs. In these studies, we demonstrate that a <em>20</em>-amino-acid peptide (EB, for entry blocker) derived from the signal sequence of <em>fibroblast</em> <em>growth</em> <em>factor</em> 4 exhibits broad-spectrum antiviral activity against influenza viruses including the H5N1 subtype in vitro. The EB peptide was protective in vivo, even when administered postinfection. Mechanistically, the EB peptide inhibits the attachment to the cellular receptor, preventing infection. Further studies demonstrated that the EB peptide specifically binds to the viral hemagglutinin protein. This novel peptide has potential value as a reagent to study virus attachment and as a future therapeutic.

Because of their ability to proliferate and to differentiate into diverse cell types, embryonic stem (ES) cells are a potential source of cells for transplantation therapy of various diseases, including Parkinson's disease. A critical issue for this potential therapy is the elimination of undifferentiated cells that, even in low numbers, could result in teratoma formation in the host brain. We hypothesize that an efficient solution would consist of purifying the desired cell types, such as neural precursors, prior to transplantation. To test this hypothesis, we differentiated sox1-green fluorescent protein (GFP) knock-in ES cells in vitro, purified neural precursor cells by fluorescence-activated cell sorting (FACS), and characterized the purified cells in vitro as well as in vivo. Immunocytofluorescence and RT-PCR analyses showed that this genetic purification procedure efficiently removed undifferentiated pluripotent stem cells. Furthermore, when differentiated into mature neurons in vitro, the purified GFP+ cell population generated enriched neuronal populations, whereas the GFP- population generated much fewer neurons. When treated with dopaminergic inducing signals such as sonic hedgehog (SHH) and <em>fibroblast</em> <em>growth</em> <em>factor</em>-8 (FGF8), FACS-purified neural precursor cells responded to these molecules and generated dopaminergic neurons as well as other neural subtypes. When transplanted, the GFP+ cell population generated well contained grafts containing dopaminergic neurons, whereas the GFP- population generated significantly larger grafts (about <em>20</em>-fold) and frequent tumor-related deaths in the transplanted animals. Taken together, our results demonstrate that genetic purification of neural precursor cells using FACS isolation can effectively remove unwanted proliferating cell types and avoid tumor formation after transplantation.

An endothelial cell <em>growth</em> <em>factor</em> has been purified about 1,000,000-fold to homogeneity from human platelets by a seven-step procedure. The purified product has an apparent Mr on sodium dodecyl sulfate-polyacrylamide gels of 45,000. The mobility in sodium dodecyl sulfate gel electrophoresis was similar in the presence or absence of reducing agents, indicating that the <em>factor</em> consists of a single polypeptide chain. Maximal stimulation by the purified protein was achieved at a concentration of about <em>20</em> ng/ml (440 pM). Heparin did not potentiate the activity, nor did the <em>factor</em> bind to heparin immobilized on Sepharose. The purified <em>factor</em> was heat- and acid-labile; it was active on porcine and human endothelial cells, but not on human foreskin <em>fibroblasts</em>. Chromatofocusing revealed that the pI of the <em>factor</em> was 4.6. The structural and functional characteristics of the platelet-derived endothelial cell <em>growth</em> <em>factor</em> are distinct from previously characterized endothelial cell mitogens with affinities for heparin.

Hypoxia is thought to be a stimulus for the excessive proliferation of vascular smooth muscle cells (VSMC) that contributes to pulmonary hypertension, but the mechanisms involved are unknown. Here we tested whether hypoxia-inducible <em>factor</em> 1-alpha (HIF-1alpha), a master regulator of the transcriptional response to hypoxia, is involved in the enhanced mitogen-induced proliferative responses of hypoxic VSMC. Exposure to moderate hypoxia (5% O(2)) enhanced the proliferative responses of human pulmonary artery SMC (HPASMC) to mitogens including platelet-derived <em>growth</em> <em>factor</em> (PDGF), <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF-2), and epidermal <em>growth</em> <em>factor</em> (EGF), compared with those in normoxia (<em>20</em>% O(2)). Moderate hypoxia elicited increased cellular HIF-1alpha levels, shown by Western blot analysis, and also enhanced PDGF-, FGF-2-, and EGF-induced expression of HIF-1alpha. Knockdown of HIF-1alpha or HIF-1beta levels in HPASMC with specific small interfering RNAs inhibited FGF-2-stimulated proliferation of HPASMC incubated in either 5% or <em>20</em>% O(2) but failed to inhibit the comitogenic effect of hypoxia. Knockdown of HIF-1alpha similarly inhibited PDGF-stimulated proliferation, whereas HIF-2alpha knockdown had no effect on HPASMC proliferation. Knockdown of HIF-1alpha expression also inhibited <em>growth</em> <em>factor</em>-induced expression of cyclin A. We conclude that HIF-1alpha promotes proliferative responses of human VSMC to FGF-2, PDGF, and EGF by mechanisms that may involve HIF-1-dependent expression of cyclin A, but HIF is apparently not crucial to the enhancement of FGF-2-, PDGF-, and EGF-induced proliferation of VSMC that occurs during hypoxia.

OBJECTIVE

To compare cell cycle kinetics in corneal endothelial cells from young and old donors.

METHODS

Human corneas were obtained from the eye bank and separated into two groups: young (19 corneas, <30 years of age) and old (40 corneas, >50 years of age). Corneas were cut in quarters, and the endothelium was released from contact inhibition by producing a 2-mm scrape wound. Unwounded endothelium acted as a negative control. Corneal pieces were exposed for 24, 36, 48, 60, 72, and 84 hours to medium containing 10% fetal bovine serum, <em>20</em> ng/ml <em>fibroblast</em> <em>growth</em> <em>factor</em>, and 50 mg/ml gentamicin or the same medium supplemented with 10 ng/ml epidermal <em>growth</em> <em>factor</em> (EGF). Tissue was fixed, immunostained for Ki67 (a marker for the late G1-through M-phase) or for 5-bromo-2'-deoxyuridine (BrdU; a marker for the S-phase), and mounted in medium containing propidium iodide (PI) to visualize all nuclei. Confocal images were evaluated using an image analysis program to count Ki67-positive and PI-stained cells and to evaluate cell cycle position. Cells were counted in 15x100 microm2 areas randomly selected from each wound, and the mean was used for subsequent calculations.

RESULTS

Human corneal endothelial cells could be reliably scored for their position within the cell cycle using Ki67 staining patterns. In both age groups, cells repopulating the wound area stained positively for Ki67, whereas no Ki67 staining was observed in unwounded areas under any condition tested. Cells from old donors treated with fetal bovine serum and FGF stained positively for Ki67, indicating that these cells were actively cycling. Compared with cells from young donors, old cells entered the cell cycle more slowly (48 versus 36 hours), the peak of Ki67 staining occurred later (72 versus 60 hours), and fewer cells proliferated (23% versus 47%) or exhibited mitotic figures (4% versus 7%). Addition of EGF to the culture medium increased Ki67 staining in both groups, but the effect on old cells was more dramatic. More cells from old donors entered the cell cycle by 36 hours after wounding, the number of proliferating cells increased 1.6-fold, and the relative number of mitotic figures increased 2.5-fold over cells treated in the absence of EGF.

CONCLUSIONS

Regardless of donor age, corneal endothelial cells can enter and complete the cell cycle. In the presence of fetal bovine serum and FGF, cells from old donors can proliferate but respond more slowly and to a lesser extent than cells from young donors. EGF added to the medium stimulates cells from old donors to enter the cell cycle faster, increases the relative number of actively cycling cells, and increases the number of cells exhibiting mitotic figures. The resultant hypothesis is that it is possible to stimulate a significant proliferative response in corneal endothelial cells from old individuals. Administration of an optimal combination of stimulatory growth factors is required under conditions in which cells have been transiently released from contact inhibition.

Human embryonic stem cells are pluripotent cells that can serve as a cell source for transplantation medicine, and as a tool to study human embryogenesis. We investigate here the potential of human embryonic stem cells to differentiate into hepatic cells. We have characterized the expression level of liver-enriched genes in undifferentiated and differentiated human embryonic stem cells by DNA microarrays. Our analysis revealed a subset of fetal hepatic enriched genes that are expressed in human embryonic stem cells upon differentiation into embryoid bodies. In order to isolate the hepatic-like cells, we introduced a reporter gene regulated by a hepatocyte-specific promoter into human embryonic stem cells. We isolated clones of human embryonic stem cells that express enhanced green fluorescent protein upon in vitro differentiation. Through immunostaining, we showed that most of these cells express albumin, while some cells still express the earlier expressed protein alpha-fetoprotein. Using fluorescence activated cell sorter, we were able to sort out the fluorescent differentiated cells and expand them for a few more weeks. This is the first report to demonstrate the possibility of purifying differentiated derivatives of human embryonic stem cells and culturing them further. Through confocal microscopy, we detected clusters of hepatic-like cells in <em>20</em>-day-old embryoid bodies and in teratomas. As observed during embryonic development, we showed that in teratomas, the hepatic-like endodermal cells develop next to cardiac mesodermal cells. In order to examine the secreted <em>factors</em> involved in the induction of hepatic differentiation, human embryonic stem cells were grown in the presence of various <em>growth</em> <em>factors</em>, demonstrating the potential involvement of acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> in the differentiation. In conclusion, given certain <em>growth</em> conditions and genetic manipulation, we can now differentiate and isolate hepatic-like cells from human embryonic stem cells.

Heart valve replacements composed of living tissue that can adapt, repair, and grow with a patient would provide a more clinically beneficial option than current inert replacements. Bioartificial valves were produced by entrapping human dermal <em>fibroblasts</em> within a fibrin gel. Using a mold design that presents appropriate mechanical constraints to the cell-induced fibrin gel compaction, gross fiber alignment (commissure-to-commissure alignment in the leaflets and circumferential alignment in the root) and the basic geometry of a native aortic valve were obtained. After static incubation on the mold in complete medium supplemented with transforming <em>growth</em> <em>factor</em> beta 1, insulin, and ascorbate, collagen fibers produced by the entrapped cells were found to coalign with the fibrin based on histological analyses. The resultant tensile mechanical properties were anisotropic. Ultimate tensile strength and tensile modulus of the leaflets in the commissural direction were 0.53 and 2.34 MPa, respectively. The constructs were capable of withstanding backpressure commensurate with porcine aortic valves in regurgitation tests (330 mmHg) and opened and closed under physiological pressure swings of 10 and <em>20</em> mmHg, respectively. These data support proof of principle of using cell-remodeled fibrin gel to produce tissue-engineered valve replacements.

The study of the development of the fetal membranes is an ancient one, and the importance of placental vascular development to placental function has long been recognized. Animal models have been important in these studies, as they allow for controlled experiments and analysis of multiple time-points during pregnancy. Since the demonstration nearly <em>20</em> years ago that the placenta produces angiogenic <em>factors</em>, the major <em>factors</em> regulating placental angiogenesis have been identified. These major <em>factors</em> include vascular endothelial <em>growth</em> <em>factor</em> (VEGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), the angiopoietins (ANG), and their receptors. Recently, sophisticated computerized image analysis methods have been developed to establish the pattern of placental vascular development in sheep. The maternal placental capillary bed develops primarily by increased size of capillaries, with only small increases in capillary number or surface densities. In contrast, the microvasculature of the fetal placenta develops primarily by increased branching, resulting in a large increase in capillary number and surface densities. These observations help to explain the relatively large increase in umbilical blood flow and nutrient delivery to the fetus that occurs during the last half of gestation. In addition, expression of mRNAs for VEGF, bFGF, ANG, and their receptors have recently been correlated with normal placental vascular development in sheep, and further refinement of these mathematical models is warranted. Lastly, the recent development of animal models of compromised pregnancies, including those resulting from maternal nutrition (both restriction and excess), multiple fetuses, environmental stress (heat stress and high altitude), and fetal and maternal breed effects, has already indicated that reductions in placental vascular development and expression of angiogenic <em>factors</em> are probably a root cause of fetal <em>growth</em> restriction. With these methods and models now in place, we should soon be able to establish the mechanisms involved in both normal and abnormal placental angiogenesis.

Uterine <em>growth</em> <em>factors</em> are potential effector molecules in embryo <em>growth</em> signaling pathways. Pig uterine luminal flushings contained a heparin-binding <em>growth</em> <em>factor</em> (HBGF) that required 0.8 M NaCl for elution from heparin columns and was termed HBGF-0.8. This <em>factor</em>, which was heat- and acid-labile and of Mr 10,000 as assessed by gel filtration, stimulated DNA synthesis in <em>fibroblasts</em> and smooth muscle cells but not endothelial cells. Two forms of HBGF-0.8, termed HBGF-0.8-P1 and HBGF-0.8-P2, exhibited differential heparin-binding properties. SDS-polyacrylamide gel electrophoresis showed that each form of HBGF-0.8 migrated with an apparent Mr of 10, 000 under reducing conditions. Amino acid sequencing revealed the N-terminal sequence EENIKKGKKXIRTPKI for HBGF-0.8-P1 and ENIKKGKKXIRT for HBGF-0.8-P2. These sequences corresponded, respectively, to residues 247-262 and 248-259 of the 349-residue predicted primary translation product of porcine connective tissue <em>growth</em> <em>factor</em> (pCTGF). 10-kDa CTGF-mediated <em>fibroblast</em> DNA synthesis was modulated by exogenous heparin, and CTGF-immunoreactive proteins of 10, 16, and <em>20</em> kDa were present in unfractionated uterine luminal flushings. These data reveal the identity of a novel <em>growth</em> <em>factor</em> in uterine fluids as a highly truncated form of CTGF and show that the N-terminal two-thirds of the CTGF primary translation product is not required for mitogenic activity or heparin binding.

We assessed the ability of acidic and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) to stimulate tyrosine kinase activity in intact cells. Immunoblot with polyclonal antiphosphotyrosine antibodies detected a 90-kDa phosphotyrosine-bearing protein in lysates of Swiss 3T3 cells exposed to pituitary-derived FGF, recombinant acidic FGF, or recombinant basic FGF, but not from unstimulated cells or cells exposed to epidermal <em>growth</em> <em>factor</em> or platelet-derived <em>growth</em> <em>factor</em>. Phosphotyrosine and its analogue phenyl phosphate, but not phosphoserine, phosphothreonine, or tyrosine itself, blocked recognition of the 90-kDa protein by antiphosphotyrosine antiserum. A monoclonal antiphosphotyrosine antibody also recognized the 90-kDa protein and was used to partially purify the protein by immunoaffinity chromatography. Phosphoamino acid analysis of the 90 kDa band revealed that it contained <em>20</em>% phosphotyrosine, 35% phosphothreonine, and 45% phosphoserine. Tyrosine phosphorylation of the 90-kDa protein was detectable within 30 s and reached a plateau within 10 min of FGF addition. The addition of suramin, which blocks the interaction of FGF with its receptor, caused rapid disappearance of the 90 kDa band. Cell fractionation experiments were consistent with the 90-kDa protein being membrane-associated, but cross-linking studies revealed that the FGF receptor had an Mr between 145 and 210 kDa in Swiss 3T3 cells, distinct from the 90-kDa major substrate for tyrosine phosphorylation. These data demonstrate that both acidic and basic FGF activate a tyrosine kinase in vivo leading to phosphorylation of a unique 90-kDa substrate, and they suggest that protein modification by phosphorylation at tyrosine is involved in eliciting the mitogenic effect of FGF.

We studied the effect of cyclic mechanical stretching on the proliferation and collagen mRNA expression and protein production of human patellar tendon <em>fibroblasts</em> under serum-free conditions. The role of transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1) in collagen production by cyclically stretched tendon <em>fibroblasts</em> was also investigated. The tendon <em>fibroblasts</em> were grown in microgrooved silicone dishes, where the cells were highly elongated and aligned with the microgrooves. Cyclic uniaxial stretching with constant frequency and duration (0.5 Hz, 4 h) but varying magnitude of stretch (no stretch, 4%, and 8%) was applied to the silicone dishes. Following the period of stretching, the cells were rested for <em>20</em> h in stretching-conditioned medium to allow for cell proliferation. In separate experiments, the cells were stretched for 4h and then rested for another 4 h. Samples of the medium, total cellular RNA and protein were used for analysis of collagen and TGF-beta1 gene expression and production. It was found that there was a slight increase in <em>fibroblast</em> proliferation at 4% and 8% stretch, compared to that of non-stretched <em>fibroblasts</em>, where at 8% stretch the increase was significant. It was also found that the gene expression and protein production of collagen type I and TGF-beta1 increased in a stretching-magnitude-dependent manner. And, levels of collagen type III were not changed, despite gene expression levels of the protein being slightly increased. Furthermore, the exogenous addition of anti-TGF-beta1 antibody eliminated the increase in collagen type I production under cyclic uniaxial stretching conditions. The results suggest that mechanical stretching can modulate proliferation of human tendon <em>fibroblasts</em> in the absence of serum and increase the cellular production of collagen type I, which is at least in part mediated by TGF-beta1.

Carbon nanotubes (CNTs) are novel materials with unique electronic and mechanical properties. The extremely small size, fiberlike shape, large surface area, and unique surface chemistry render their distinctive chemical and physical characteristics and raise potential hazards to humans. Several reports have shown that pulmonary exposure to CNTs caused inflammation and lung fibrosis in rodents. The molecular mechanisms that govern CNT lung toxicity remain largely unaddressed. Here, we report that multiwalled carbon nanotubes (MWCNTs) have potent, dose-dependent toxicity on cultured human lung cells (BEAS-2B, A549, and WI38-VA13). Mechanistic analyses were carried out at subtoxic doses (≤<em>20</em> μg/mL, ≤ 24 h). MWCNTs induced substantial ROS production and mitochondrial damage, implicating oxidative stress in cellular damage by MWCNT. MWCNTs activated the NF-κB signaling pathway in macrophages (RAW264.7) to increase the secretion of a panel of cytokines and chemokines (TNFα, IL-1β, IL-6, IL-10, and MCP1) that promote inflammation. Activation of NF-κB involved rapid degradation of IκBα, nuclear accumulation of NF-κBp65, binding of NF-κB to specific DNA-binding sequences, and transactivation of target gene promoters. Finally, MWCNTs induced the production of profibrogenic <em>growth</em> <em>factors</em> TGFβ1 and PDGF from macrophages that function as paracrine signals to promote the transformation of lung <em>fibroblasts</em> (WI38-VA13) into myo<em>fibroblasts</em>, a key step in the development of fibrosis. Our results revealed that MWCNTs elicit multiple and intertwining signaling events involving oxidative damage, inflammatory cytokine production, and myofibroblast transformation, which potentially underlie the toxicity and fibrosis in human lungs by MWCNTs.

The present study compares some phenotypic and physiologic characteristics of microvascular and macrovascular endothelial cells from within one human organ. To this end microvascular endothelial cells from human full-term placenta (PLEC) were isolated using a new method and compared with macrovascular human umbilical vein endothelial cells (HUVEC) and an SV40-transformed placental venous endothelial cell line (HPEC-A2). PLEC were isolated by enzymatic perfusion of small placental vessels, purified on a density gradient and cultured subsequently. Histological sections of the enzyme-treated vessels showed a selective removal of the endothelial lining in the perfused placental cotyledons. The endothelial identity of the cells was confirmed by staining with the endothelial markers anti-von Willebrand <em>factor</em>, Ulex europaeus lectin and anti-QBEND10. The cells internalized acetylated low-density lipoprotein and did not show immunoreactivity with markers for macrophages, smooth muscle cells and <em>fibroblasts</em>. The spindle-shaped PLEC grew in swirling patterns similar to that described for venous placental endothelial cells. However, scanning electron microscopic examination clearly showed that PLEC remained elongated at the confluent state, in contrast to the more polygonal phenotype of HPEC-A2 and HUVEC that were studied in parallel. The amount of vasoactive substances (endothelin-1,2, thromboxane, angiotensin II, prostacyclin) released into the culture medium and the proliferative response to cytokines was more similar to human dermal microvessels (MIEC) derived from non-fetal tissue than to HUVEC. Potent mitogens such as vascular endothelial <em>growth</em> <em>factors</em> (VEGF121, VEGF165) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) induced proliferation of all endothelial cell types. Placental <em>growth</em> <em>factors</em> PIGF-1 and PIGF-2 effectively stimulated cell proliferation on PLEC (142 +/- 7% and 173 +/- 10%) and MIEC (160 +/- <em>20</em>% and 143 +/- 28%) in contrast to HUVEC (9 +/- 8% and 15 +/- <em>20</em>%) and HPEC-A2 (15 +/- 7% and 24 +/- 6%) after 48 h incubation time under serum-free conditions. These data support evidence for (1) the microvascular identity of the isolated PLEC described in this study, and (2) the phenotypic and physiologic heterogeneity of micro- and macrovascular endothelial cells within one human organ.

The pathogenic process responsible for the loss of dopaminergic neurons within the substantia nigra of patients with Parkinson disease (PD) is poorly understood. Current research supports the involvement of fibroblast growth factor (FGF20) in the survival of dopaminergic cells. FGF20 is a neurotrophic factor that is preferentially expressed within the substantia nigra of rat brain. The human homologue has been mapped to 8p21.3-8p22, which is within an area of PD linkage revealed through our published genomic screen. To test whether FGF20 influences risk of PD, we genotyped five single-nucleotide polymorphisms (SNPs) lying within the FGF20 gene, in a large family study. We analyzed our sample (644 families) through use of the pedigree disequilibrium test (PDT), the genotype PDT, the multilocus-genotype PDT, and the family-based association test to assess association between risk of PD and alleles, genotypes, multilocus genotypes, and haplotypes. We discovered a highly significant association of PD with one intronic SNP, rs1989754 (P=.0006), and two SNPs, rs1721100 (P=.02) and ss20399075 (P=.0008), located in the 3' regulatory region in our overall sample. Furthermore, we detected a haplotype (A-G-C-C-T) that is positively associated with risk of PD (P=.0003), whereas a second haplotype (A-G-G-G-C) was found to be negatively associated with risk of PD (P=.0009). Our results strongly support FGF20 as a risk factor for PD.

BACKGROUND

Blood flow is impaired in islet transplants, but there is conflicting evidence on improving the outcome by promoting vascularization. We previously reported that islet endothelial cells (EC) possess significant angiogenic capacity.

METHODS

To further address this issue, we studied human islets in culture under hypoxic conditions. Moreover, we used a transgene mouse model with human vascular endothelial growth factor (VEGF) production in beta-cells under the control of the rat insulin promoter (RIP) to stimulate islet EC proliferation.

RESULTS

Subsequent to a hypoxic stimulus, islets responded with specific expression patterns of VEGF and fibroblast growth factor; however, this was not sufficient to prevent the decay of islet EC. VEGF release of RIP-VEGF transgenic islets was controlled by glucose and resulted in the formation of sprouts. When transplanted to the kidney capsule of diabetic mice, RIP-VEGF islets significantly enhanced microvascular density and functional blood flow to the graft compared with controls.

CONCLUSIONS

Optimized angiogenesis of islet transplants resulted in greater availability of insulin caused by beta-cell proliferation and a significantly higher percentage (90% versus 20%) of mice cured from diabetes.

Aberrant angiogenesis is an essential step for the progression of solid tumors. Thus anti-angiogenic therapy is one of the most promising approaches to control tumor <em>growth</em>. In this study, we examined the ability of <em>20</em>(R)-ginsenoside Rg3 (Rg3), one of the active compounds present in ginseng root, to interfere with the various steps of angiogenesis. Rg3 was found to inhibit the proliferation of human umbilical vein endothelial cells (HUVEC) with an IC50 of 10 nM in Trypan blue exclusion assay. Rg3 (1-10(3) nM) also dose dependently suppressed the capillary tube formation of HUVEC on the Matrigel in the presence or absence of <em>20</em> ng/ml vascular endothelial <em>growth</em> <em>factor</em> (VEGF). The VEGF-induced chemoinvasion of HUVEC and ex vivo microvascular sprouting in rat aortic ring assay were both significantly attenuated by Rg3. In addition, Rg3 (150 and 600 nM) remarkably abolished the basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF)-induced angiogenesis in an in vivo Matrigel plug assay. The Matrix metalloproteinases (MMPs), such as MMP-2 and MMP-9, which play an important role in the degradation of basement membrane in angiogenesis and tumor metastasis present in the culture supernatant of Rg3-treated aortic ring culture were found to decrease in their gelatinolytic activities. Taken together, these data underpin the anti-tumor property of Rg3 through its angiosuppressive activity.