Angiogenesis plays a key role in solid tumor <em>growth</em>. The purpose of this work was to study angiogenesis in acute myeloid leukemia (AML). We stained bone marrow samples from <em>20</em> adult patients with untreated AML and <em>20</em> normal controls using endothelial cell markers (ULEX-E and von Willebrand <em>factor</em> [vWF]). The number of vessels per millimeter length of bone marrow core biopsy specimen was scored by light microscopy. Using ULEX-E staining, AML marrows had (average +/- SEM) 8.3 +/- 3.6 vessels/mm (range, 3.7-19.3), whereas normal marrows had 4.3 +/- 1.8 vessels/mm (range, 1.6-7.9). A similar difference was noted using vWF staining (8.6 +/- 3.0 vessels/mm vs 4. 9 +/- 2.2 vessels/mm in AML vs normal bone marrows, respectively). The differences between the numbers of vessels/mm in AML and normal marrows were highly significant (P <.0001 for both ULEX-E and vWF staining). When analyzed by FAB category, there was no difference in the average number of vessels/mm among the different subgroups of AML. Using reverse transcriptase polymerase chain reaction, we observed that the HL-60 and U937 human AML cell lines and 4 of 4 freshly isolated AML cells from untreated patients expressed mRNA for vascular endothelial <em>growth</em> <em>factor</em> (VEGF). Both cell lines as well as all fresh AML isolates tested expressed VEGF protein. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> was expressed only in HL-60 cells and in only 3 of 4 fresh AML samples. These observations suggest that angiogenesis may play a role in the pathogenesis of AML. Inhibition of angiogenesis could constitute a novel strategy for the treatment of AML. (Blood. <em>20</em>00;95:309-313).

Most endometrial carcinomas are diagnosed at an early stage. Still, 15-<em>20</em>% of these carcinomas recur with limited effect of systemic therapies in metastatic disease. Improved ability to target surgical and systemic therapies to well selected patient populations will increase the likelihood of benefits. Retrospective studies have identified several markers for lymph-node metastasis and poor prognosis. No new targeted treatments are available in the clinic, but recent comprehensive molecular characterisations of tumours have identified drugs targeting the PI3K/PTEN/AKT/mTOR pathway and <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) 2 as promising for further studies, also reflected in current clinical trials investigating endometrial carcinoma. A more systematic approach to integration of biomarkers in surgical trials and clinical trials of therapeutics, earlier characterisation and standardisation of diagnostic imaging and biomarker assessment, and prospective implementation studies are needed for clinical implementation. We summarise the present knowledge regarding biomarkers in endometrial carcinoma, assessing how such markers could be applied to address key clinical challenges for the treatment of this disease.

Transforming <em>growth</em> <em>factors</em> of the beta-class (TGFs-beta) stimulate extracellular matrix synthesis and have been implicated in embryogenesis, wound healing, and fibroproliferative responses to tissue injury. Because cells communicate with several extracellular matrix components via specific cell membrane receptors, we hypothesized that TGFs-beta may also regulate the expression of such receptors. We confirmed that TGF-beta 1 increases the expression of fibronectin, an adhesive glycoprotein expressed during embryogenesis and tissue remodeling. Based upon the 48-72-h period required for a maximal fibroproliferative response to dermal injections of TGF-beta 1, we exposed human fetal lung <em>fibroblasts</em> (IMR-90) to TGF-beta 1 for periods up to 48 h in vitro. We observed as much as 6-fold increases in fibronectin synthesis by 24 h as previously reported for fibroblastic cells (Ignotz, R. A., and Massagué, J. (1986) J. Biol. Chem. 261, 4337-4345; Ignotz, R. A., Endo, T., and Massagué, J. (1987) J. Biol. Chem. 262, 6443-6446; Raghow, R., Postlethwaithe, A. E., Keski-Oja, J., Moses, H. L., and Kang, A. H. (1987) J. Clin. Invest. 79, 1285-1288), but up to 30-fold increases by 48 h. These increases are accompanied by similar increases in fibronectin mRNA levels which are prevented by actinomycin D treatment. Using a monospecific antibody raised to the human placental fibronectin receptor complex, we found that TGF-beta 1 stimulated fibronectin receptor synthesis up to <em>20</em>-40-fold and increases mRNA levels encoding both the alpha- and beta-subunits up to 3-fold, compared to control IMR-90 in serum-free medium. Actinomycin D blocks TGF-beta 1-mediated increases in receptor mRNA levels. The earliest detectable TGF-beta 1-mediated increases in fibronectin receptor complex protein synthesis and mRNA levels occur at 8 h, whereas the earliest increases in fibronectin protein synthesis and mRNA levels occur at 12 h. These results demonstrate that TGF-beta 1 stimulates fibronectin receptor synthesis, extending the diverse stimulatory activities of this polypeptide to matrix receptors. In addition, because fibronectin matrix assembly may involve the fibronectin cell adhesive receptor complex, increased receptor expression may help drive fibronectin deposition into matrix.

BACKGROUND

The value of these prognostic factors was compared with that of other clinicopathologic factors such as tumor grade, tumor stage, mucin production, vascular invasion, perineural invasion, and lymphatic invasion.

OBJECTIVE

To determine whether the development of distant recurrence in patients with node-negative colon cancer could be predicted using vessel count and vascular endothelial growth factor (VEGF) expression.

METHODS

Paraffin-embedded colon cancers were immunostained for factor VIII, VEGF, basic fibroblast growth factor, and proliferating cell nuclear antigen; slides were reviewed for differentiation, mucin production, and the presence of vascular, lymphatic, and/or perineural invasion.

METHODS

A large academic cancer referral center where 27 patients with node-negative colon cancer were operated on during 1988 and 1989.

METHODS

The development of and interval to recurrence.

RESULTS

Eight patients developed liver, lung, or lymph node metastases at a median of 24 months. The median follow-up for patients without cancer recurrence was 60 months. The mean tumor vessel count for those patients who remained disease-free was significantly fewer than for those patients who suffered a recurrence (20 vs 33, respectively). By univariate analysis, 3 factors- perineural invasion, vessel count, and VEGF expression- were correlated with time to recurrence. By multivariate analysis, only vessel count was significantly related to differences in time to recurrence. Expression of VEGF correlated with vessel count.

CONCLUSIONS

Vessel count and expression of VEGF may be useful for predicting distant recurrence in patients with node-negative colon cancer.

OBJECTIVE

The clinical use of human mesenchymal stromal cells (MSC) requires ex vivo expansion in media containing supplements such as fetal bovine serum or, alternatively, human platelet lysate (PL).

METHODS

Platelet concentrates were frozen, quarantine stored, thawed and sterile filtered to obtain PL. PL content and its effect on fibroblast-colony-forming unit (CFU-F) formation, MSC proliferation and large-scale expansion were studied.

RESULTS

PL contained high levels of basic fibroblast growth factor (bFGF), soluble CD40L (sCD40L), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), platelet-derived growth factor AA (PDGF-AA), platelet-derived growth factor AB/BB (PDGF-AB/BB), chemokine (C-C) ligand 5 (CCL5; RANTES) transforming growth factor-β1 (TGF-β1) and chemokine (C-X-C) ligand 1/2/3 (GRO), with low batch-to-batch variability, and most were stable for up to 14 days. Inhibition of PDGF-BB and bFGF decreased MSC proliferation by about 20% and 50%, respectively. The strongest inhibition (about 75%) was observed with a combination of anti-bFGF + anti-PDGF-BB and anti-bFGF + anti-TGF-β1 + anti-PDGF-BB. Interestingly, various combinations of recombinant PDGF-BB, bFGF and TGF-β1 were not sufficient to promote cell proliferation. PL from whole blood-derived pooled platelet concentrates and apheresis platelet concentrates did not differ significantly in their growth-promoting activity on MSC.

CONCLUSIONS

PL enhances MSC proliferation and can be regarded as a safe tool for MSC expansion for clinical purposes. \in particular, PDGF-BB and bFGF are essential components for the growth-promoting effect of PL, but are not sufficient for MSC proliferation.

OBJECTIVE

Keratocytes give rise to fibroblasts and myofibroblasts in wounded cornea. It is well established that treatment of fibroblasts with transforming growth factor (TGF) beta will induce myofibroblast differentiation. We investigated whether this differentiation could be reversed by the administration of fibroblast growth factor (FGF).

METHODS

Cultured corneal myofibroblasts were plated at 200 cells/mm(2), and cells were grown in DMEM/F12 containing (1) 10% FBS or (2) 10% FBS with FGF and heparin or (3) 1% FBS or (4) 1% FBS with TGF-beta. As distinguished from the fibroblast phenotype, the myofibroblast phenotype was identified by the assembly of alpha-smooth muscle (SM) actin protein into the stress fiber cytoskeleton. To further characterize growth factor regulation of the two phenotypes, the phenotypic expression of TGF-beta receptor types I and II, cadherins, and connexin 43 by immunocytochemistry, Western blot analysis, and immunoprecipitation and of alpha-SM actin mRNA in Northern blot analysis were evaluated.

RESULTS

Corneal myofibroblasts replated and grown in the presence of FGF-1 or FGF-2 (20 ng/ml) plus heparin (5 microg/ml) in 10% FBS medium had decreased expression of alpha-SM actin protein, TGF-beta receptors, and cadherins. Thus, FGF-heparin decreased the myofibroblast phenotype and promoted the fibroblast phenotype. Administration of either 20 ng/ml FGF or 5 microg/ml heparin alone was not effective. Addition of TGF-beta further enhanced the expression of alpha-SM actin mRNA and protein and cell surface expression of TGF-beta receptors in myofibroblast cultures.

CONCLUSIONS

FGF-1 or -2 and heparin promoted the fibroblast phenotype and reversed the myofibroblast phenotype. This finding supports the idea that corneal myofibroblasts and fibroblasts are alternative phenotypes rather than terminally differentiated cell types.

Transforming <em>growth</em> <em>factor</em> beta (TGF-beta) increases up to <em>20</em>-fold the expression of various forms of chondroitin/dermatan sulfate proteoglycan, the major type of sulfated proteoglycan present in the extracellular matrix and culture medium of various human, rodent, and mink cell types including kidney and lung <em>fibroblasts</em>, lung epithelial cells, preadipocytes, and skeletal muscle myoblasts. TGF-beta regulates the level and molecular size of these proteoglycans by acting simultaneously at two levels: it elevates the biosynthetic rate of the 45-kDa proteoglycan core protein in a cycloheximide- and actinomycin D-sensitive manner, and it induces an increase in the molecular mass of the glycosaminoglycan chains. These cellular responses correlate with occupancy of type III TGF-beta receptors by TGF-beta 1 and TGF-beta 2 and are not induced by other <em>growth</em> <em>factors</em> tested. The parameters of this effect of TGF-beta in kidney <em>fibroblasts</em> and myoblasts are ED50 = 5-10 pM TGF-beta 1 or TGF-beta 2, and t 1/2 = 6-8 h. These results identify the chondroitin/dermatan sulfate proteoglycans as a major component of mammalian mesenchymal and epithelial extracellular matrices whose expression and structure are regulated by TGF-beta.

Angiogenesis plays a key role in tumor <em>growth</em> and metastasis. The transforming <em>growth</em> <em>factor</em> alpha (TGF-alpha)-epidermal <em>growth</em> <em>factor</em> receptor (EGFR) autocrine pathway controls in part the production of angiogenic <em>factors</em> such as vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) in cancer cells. In this study, we have evaluated the antiangiogenic and antitumor activity of monoclonal antibody (MAb) C225, an anti-EGFR chimeric human-mouse MAb, alone and in combination with a human VEGF antisense (AS) 21-mer phosphorothioate oligonucleotide (VEGF-AS) in human GEO colon cancer cells. MAb C225 treatment determined a dose-dependent inhibition of VEGF, bFGF, and TGF-alpha production by GEO cells in vitro. Treatment with VEGF-AS caused a selective inhibition in VEGF expression by GEO cells in vitro. Treatment of immunodeficient mice bearing established, palpable GEO xenografts for 3 weeks with VEGF-AS or with MAb C225 determined a cytostatic reversible inhibition of tumor <em>growth</em>. In contrast, a prolonged inhibition of tumor <em>growth</em> was observed in all mice treated with the two agents, in combination with a significant improvement in mice survival compared with controls (P < .001), to MAb C225 (P < .001), or to VEGF-AS (P < .001) treated mice. All mice died within 4, 6, and 8 weeks after tumor cell injection in the control, VEGF-AS and MAb C225 groups, respectively. In contrast, 50% of mice treated with the combination of VEGF-AS and MAb C225 were alive at 13 weeks. Ten % of mice treated with VEGF-AS plus MAb C225 were alive at <em>20</em> weeks and had no histological evidence of GEO tumors. Immunohistochemical analysis of GEO tumor xenografts demonstrated a significant reduction of VEGF expression after treatment with VEGF-AS with a parallel reduction in microvessel count. MAb C225 treatment determined a reduction in the expression of VEGF, bFGF, and TGF-alpha with a reduction in microvessel count. Finally, a significant potentiation in inhibition of VEGF expression and little or no microvessels were observed in GEO tumors after the combined treatment with the two agents.

Both epidermal <em>growth</em> <em>factor</em> receptor (EGF-R) signaling mechanisms and angiogenesis have been evaluated as independent targets for therapy of human pancreatic carcinoma, but a link between the two processes has been identified only recently. This study evaluated whether EGF-R blockade therapy with anti-EGF-R antibody C225 inhibits pancreatic carcinoma <em>growth</em> and metastasis in an orthotopic nude mouse model via tumor-mediated angiogenesis and whether gemcitabine potentiates this effect. In vitro treatment of human pancreatic carcinoma L3.6pl cells with C225 inhibited EGF-R autophosphorylation, producing a maximum of <em>20</em>% cytostasis. Treatment with C225 plus gemcitabine resulted in additive cytotoxic effects that increased with increasing gemcitabine concentrations. Dose-dependent decreases in expression of the angiogenic <em>factors</em> vascular endothelial <em>growth</em> <em>factor</em> and interleukin 8 (but not basic <em>fibroblast</em> <em>growth</em> <em>factor</em>) were observed in the C225-treated cells (mRNA and protein levels). In L3.6pl tumors established in the pancreas of nude mice, systemic therapy with C225 alone and C225 in combination with gemcitabine resulted in <em>growth</em> inhibition, tumor regression, and abrogation of metastasis; median tumor volume was reduced from 538 to 0.3 and to 0 mm3, respectively. Gemcitabine treatment alone reduced median tumor volume from 538 to 152 mm3. Liver metastases were present in 50% of the controls, 30% of the gemcitabine-treated animals, and <em>20</em>% of C225-treated animals. No macroscopically visible liver metastases were observed in the combination treatment group. As early as 11 days after C225 treatment, the median percentage of proliferating cell nuclear antigen-positive cells was substantially reduced compared with gemcitabine treatment alone (26% versus 73%, respectively) versus controls (92%), correlating with in vivo blockade of EGF-R activation. Similarly after 11 days treatment, production of vascular endothelial <em>growth</em> <em>factor</em> and interleukin 8 was significantly lower in C225 and C225 plus gemcitabine-treated tumors versus gemcitabine-treated and control tumors. Significant differences in microvessel density were observed 18 days after C225 or combination treatments (but not gemcitabine alone) in direct correlation with the difference in percentage of apoptotic endothelial cells, as visualized by double immunofluorescence microscopy. These experiments indicate that therapeutic strategies targeting EGF-R have a significant antitumor effect on human L3.6pl pancreatic carcinoma <em>growing</em> in nude mice which is mediated in part by inhibition of tumor-induced angiogenesis, leading to tumor cell apoptosis and regression. Furthermore, this effect is potentiated in combination with gemcitabine.

Platelet-derived <em>growth</em> <em>factor</em> (PDGF) is a potent activator for cells of mesenchymal origin. PDGF stimulates chemotaxis, proliferation, and new gene expression in monocytes-macrophages and <em>fibroblasts</em> in vitro, cell types considered essential for tissue repair. Therefore, we analyzed the influence of exogenously administered recombinant B chain homodimers of PDGF (PDGF-BB) on two experimental tissue repair paradigms, incisional and excisional wounds. In both types of wounds, as little as <em>20</em>-<em>20</em>0 picomoles applied a single time to wounds significantly augmented the time dependent influx of inflammatory cells and <em>fibroblasts</em> and accelerated provisional extracellular matrix deposition and subsequent collagen formation. In incisional wounds, PDGF-BB augmented wound breaking strength 50-70% over the first 3 weeks; in excisional wounds, PDGF-BB accelerated time to closure by 30%. PDGF-BB exaggerated, but did not alter, the normal course of soft tissue repair, resulting in a significant acceleration of healing. Long term observations established no apparent differences between PDGF-BB treated and non-treated wounds. Thus, the vulnerary effects of PDGF-BB were transient and fully reversible in both wound healing models. Furthermore, analysis of PDGF-treated and non-treated wounds has provided important insights into mechanisms of normal and deficient tissue repair processes. PDGF appears to transduce its signal through wound macrophages and may trigger the induction of positive autocrine feedback loops and synthesis of endogenous wound PDGF and other <em>growth</em> <em>factors</em>, thereby enhancing the cascade of tissue repair processes required for a fully-healed wound. Thus, PDGF and other wound produced polypeptide <em>growth</em> <em>factors</em> may be the critical regulators of extracellular matrix deposition within healing wounds.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) has been found to increase neuronal survival and neurite extension in a highly purified population of fetal rat hippocampal neurons under well-defined serum-free cell culture conditions. In the presence of FGF, neuronal survival after 7 days in culture on a simple plastic substrate is increased 4-fold, to 54% of the initial population. Survival is increased 2-fold to 40% on polyornithine-laminin. When FGF was bound to plastic or heparin substrates, neurite out<em>growth</em> was significantly increased to lengths comparable to those seen with laminin; however, FGF produced no further increase in neurite out<em>growth</em> on laminin. Half-maximal survival was observed at FGF concentrations of about 15 pg/ml (1 pM); half-maximal process out<em>growth</em> occurred at about 375 pg/ml (<em>20</em> pM). The responsive cells were identified as neurons by their labeling with tetanus toxin and by antibodies to neurofilaments and to the neuron-specific enolase. Astrocytes, identified by the presence of glial fibrillary acidic protein, constituted about 10% of cells present at 1 week both in the presence and in the absence of FGF. These results strongly suggest that, in addition to its known mitogenic effects on nonneuronal cells, FGF possesses neurotrophic activity for hippocampal neurons.

Recombinant expression of a chimeric EGFR/ErbB-3 receptor in NIH 3T3 <em>fibroblasts</em> allowed us to investigate cytoplasmic events associated with ErbB-3 signal transduction upon ligand activation. An EGFR/ErbB-3 chimera was expressed on the surface of NIH 3T3 transfectants as two classes of receptors possessing epidermal <em>growth</em> <em>factor</em> (EGF) binding affinities comparable to those of the wild-type EGF receptor (EGFR). EGF induced autophosphorylation in vivo of the chimeric receptor and DNA synthesis of EGFR/ErbB-3 transfectants with a dose response similar to that of EGFR transfectants. However, the ErbB-3 and EGFR cytoplasmic domains exhibited striking differences in their interactions with several known tyrosine kinase substrates. We demonstrated strong association of phosphatidylinositol 3-kinase activity with the chimeric receptor upon ligand activation comparable in efficiency with that of the platelet-derived <em>growth</em> <em>factor</em> receptor, while the EGFR exhibited a 10- to <em>20</em>-fold-lower efficiency in phosphatidylinositol 3-kinase recruitment. By contrast, both phospholipase C gamma and GTPase-activating protein failed to associate with or be phosphorylated by the ErbB-3 cytoplasmic domain under conditions in which they coupled with the EGFR. In addition, though certain signal transmitters, including Shc and GRB2, were recruited by both kinases, EGFR and ErbB-3 elicited tyrosine phosphorylation of distinct sets of intracellular substrates. Thus, our findings show that ligand activation of the ErbB-3 kinase triggers a cytoplasmic signaling pathway that hitherto is unique within this receptor subfamily.

Progenitor cells were isolated from the developing human central nervous system (CNS), induced to divide using a combination of epidermal <em>growth</em> <em>factor</em> and <em>fibroblast</em> <em>growth</em> <em>factor</em>-2, and then transplanted into the striatum of adult rats with unilateral dopaminergic lesions. Large grafts were found at 2 weeks survival which contained many undifferentiated cells, some of which were migrating into the host striatum. However, by <em>20</em> weeks survival, only a thin strip of cells remained at the graft core while a large number of migrating astrocytes labeled with a human-specific antibody could be seen throughout the striatum. Fully differentiated graft-derived neurons, also labeled with a human-specific antibody, were seen close to the transplant site in some animals. A number of these neurons expressed tyrosine hydroxylase and were sufficient to partially ameliorate lesion-induced behavioral deficits in two animals. These results show that expanded populations of human CNS progenitor cells maintained in a proliferative state in culture can migrate and differentiate into both neurons and astrocytes following intracerebral grafting. As such these cells may have potential for development as an alternative source of tissue for neural transplantation in degenerative diseases.

BACKGROUND

Basic fibroblast growth factor promotes angiogenesis and mitogenesis in colon carcinomas. Pituitary-tumour transforming gene (PTTG1) causes in-vitro and in-vivo transformation, regulates secretion of basic fibroblast growth factor, and inhibits chromatid separation. Most normal tissues show little or no PTTG1 expression but cancer cells express the gene abundantly. We postulated that PTTG1 expression in colorectal tumours is related to tumour invasiveness.

METHODS

PTTG1 gene and protein expression were assessed in 68 colorectal tumours and compared with invasive characteristics, such as lymph-node invasion, evidence of metastases, tumour vessel density, and expression of basic fibroblast growth factor. PTTG1 expression is given in terms of the fold-increase over that in normal-adjacent colorectal tissue.

RESULTS

PTTG1 was overexpressed in all of 48 colon carcinomas (median fold-increase 2.2 [IQR 1.8-3.3]) and in 19 of 20 colonic polyps (2.2 [1.6-3.1]) compared with normal colonic tissue. Invasion of surrounding lymph nodes was associated with higher PTTG1 expression than in carcinomas limited to the bowel wall (3.4 [2.1-5.9] vs 1.9 [1.7-2.4], p=0.007), and higher PTTG1 expression was seen in more vascular than in less vascular tumours (2.6 [1.9-5.1] vs 1.9 [1.8-2.5], p=0.04).

CONCLUSIONS

Increased tumour PTTG1 expression may be a marker of invasive colorectal carcinoma and could represent a new therapeutic target.

Since the days of Hans Spemann, the ocular lens has served as one of the most important developmental systems for elucidating fundamental processes of induction and differentiation. More recently, studies in the lens have contributed significantly to our understanding of cell cycle regulation and apoptosis. Over <em>20</em> years of accumulated evidence using several different vertebrate species has suggested that <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) and/or <em>fibroblast</em> <em>growth</em> <em>factor</em> receptors (FGFRs) play a key role in lens development. FGFR signaling has been implicated in lens induction, lens cell proliferation and survival, lens fiber differentiation and lens regeneration. Here we will review and discuss historical and recent evidence suggesting that (FGFR) signaling plays a vital and universal role in multiple aspects of lens development.

Normal chicken, mouse, and human embryo <em>fibroblasts</em> release into their culture media transforming <em>growth</em> <em>factors</em> (TGFs) in a latent form. Their soft agar colony-forming activity on two widely used target cells, rat NRK-49F and mouse AKR-2B, is essentially revealed only after prior acidification of cell-conditioned media. These TGFs are EGF-dependent when assayed on NRK-49F cells and EGF-independent on AKR-2B cells. The TGF activity from the chicken source is released in three (apparent) molecular weight forms of 500 kd, 125 kd, and <em>20</em> kd.

Birth defects, which occur in one out of <em>20</em> live births, often affect multiple organs that have common developmental origins. Human and mouse studies indicate that haploinsufficiency of the transcription <em>factor</em> TBX1 disrupts pharyngeal arch development, resulting in the cardiac and craniofacial features associated with microdeletion of 22q11 (del22q11), the most frequent human deletion syndrome. Here, we have generated an allelic series of Tbx1 deficiency that reveals a lower critical threshold for Tbx1 activity in the cardiac outflow tract compared with other pharyngeal arch derivatives, including the palatal bones. Mice hypomorphic for Tbx1 failed to activate expression of the forkhead transcription <em>factor</em> Foxa2 in the pharyngeal mesoderm, which contains cardiac outflow precursors derived from the anterior heart field. We identified a Fox-binding site upstream of Tbx1 that interacted with Foxa2 and was necessary for pharyngeal mesoderm expression of Tbx1, revealing an autoregulatory loop that may explain the increased cardiac sensitivity to Tbx1 dose. Downstream of Tbx1, we found a <em>fibroblast</em> <em>growth</em> <em>factor</em> 8 (Fgf8) enhancer that was dependent on Tbx1 in vivo for regulating expression in the cardiac outflow tract, but not in pharyngeal arches. Consistent with its role in regulating cardiac outflow tract cells Tbx1 gain of function resulted in expansion of the cardiac outflow tract segment derived from the anterior heart field as marked by Fgf10. These findings reveal a Tbx1-dependent transcriptional and signaling network in the cardiac outflow tract that renders mouse cardiovascular development more susceptible than craniofacial development to a reduction in Tbx1 dose, similar to humans with del22q11.

Hepatocellular carcinoma (HCC) is a typical hypervascular tumor. However, the relationship between the vascularity of HCC and the expression of angiogenic <em>factors</em> has not been investigated. In addition, no detailed studies have examined the possible involvement of angiogenic <em>factors</em> in the grade of malignancy of HCC. The aim of this study was to determine which angiogenic <em>factors</em> regulate tumor angiogenesis and contribute to the invasive ability of liver tumors, especially of HCC. Northern blot analysis was used to examine the transcriptional expression of vascular endothelial <em>growth</em> <em>factor</em> (VEGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF), and acidic FGF in resected surgical specimens (<em>20</em> HCC and 9 metastatic liver tumors). Correlations between messenger RNA (mRNA) expression and arteriographic findings, as well as histopathological findings, were evaluated. Immunohistochemistry was performed to identify the localization of cells expressing VEGF in HCC. Higher levels of VEGF mRNA were observed in 12 of <em>20</em> HCC and 2 of 9 metastatic liver tumors than in corresponding nontumorous tissues. The degree of VEGF mRNA expression was significantly correlated with the intensity of tumor staining in angiograms (P<.01). On immunohistochemical observation, VEGF protein was intensely detected in HCC cells. Furthermore, basic FGF mRNA was detected in 9 of <em>20</em> HCC and was related to the capsular infiltration of cancer cells (P<.05). In contrast, no significant difference was observed in the very low levels of acidic FGF mRNA found in the tumorous and nontumorous portions of the liver. In conclusion, these results suggest that VEGF contributes to angiogenesis of liver tumors, whereas basic FGF may be involved in the invasion of HCC into the surrounding tissues.

Single gold-tagged epidermal <em>growth</em> <em>factor</em> (EGF) molecules bound to cellular EGF receptors of fixed <em>fibroblast</em> cells were imaged in liquid with a scanning transmission electron microscope (STEM). The cells were placed in buffer solution in a microfluidic device with electron transparent windows inside the vacuum of the electron microscope. A spatial resolution of 4 nm and a pixel dwell time of <em>20</em> micros were obtained. The liquid layer was sufficiently thick to contain the cells with a thickness of 7 +/- 1 microm. The experimental findings are consistent with a theoretical calculation. Liquid STEM is a unique approach for imaging single molecules in whole cells with significantly improved resolution and imaging speed over existing methods.

Ex vivo expansion of hematopoietic stem cells (HSCs) is important for many clinical applications, and knowledge of the surface phenotype of ex vivo-expanded HSCs will be critical to their purification and analysis. Here, we developed a simple culture system for bone marrow (BM) HSCs using low levels of stem cell <em>factor</em> (SCF), thrombopoietin (TPO), insulin-like <em>growth</em> <em>factor</em> 2 (IGF-2), and <em>fibroblast</em> <em>growth</em> <em>factor</em>-1 (FGF-1) in serum-free medium. As measured by competitive repopulation analyses, there was a more than <em>20</em>-fold increase in numbers of long-term (LT)-HSCs after a 10-day culture of total BM cells. Culture of BM "side population" (SP) cells, a highly enriched stem cell population, for 10 days resulted in an approximate 8-fold expansion of repopulating HSCs. Similar to freshly isolated HSCs, repopulating HSCs after culture were positive for the stem cell markers Sca-1, Kit, and CD31 and receptors for IGF-2. Surprisingly, prion protein and Tie-2, which are present on freshly isolated HSCs, were not on cultured HSCs. Two other HSC markers, Endoglin and Mpl, were expressed only on a portion of cultured HSCs. Therefore, the surface phenotype of ex vivo-expanded HSCs is different from that of freshly isolated HSCs, but this plasticity of surface phenotype does not significantly alter their repopulation capability.

mRNA expression of vascular endothelial <em>growth</em> <em>factor</em> (VEGF), <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2), and hypoxia-inducible <em>factor</em> (HIF) subunits HIF-1alpha and HIF-1beta in human skeletal muscle was studied during endurance exercise at different degrees of oxygen delivery. Muscle biopsies were taken before and after 45 min of one-legged knee-extension exercise performed under conditions of nonrestricted or restricted blood flow (approximately 15-<em>20</em>% lower) at the same absolute workload. Exercise increased VEGF mRNA expression by 178% and HIF-1beta by 340%, but not HIF-1alpha and FGF-2. No significant differences between the restricted and nonrestricted groups were observed. The exercise-induced increase in VEGF mRNA was correlated to the exercise changes in HIF-1alpha and HIF-1beta mRNA. The changes in VEGF, HIF-1alpha, and HIF-1beta mRNAs were correlated to the exercise-induced increase in femoral venous plasma lactate concentration. It is concluded that 1) VEGF but not FGF-2 gene expression is upregulated in human skeletal muscle by a single bout of dynamic exercise and that there is a graded response in VEGF mRNA expression related to the metabolic stress and 2) the increase in VEGF mRNA expression correlates to the changes in both HIF-1alpha and HIF-1beta mRNA.

A series of nontransformed human and murine cells and derivative cell lines transformed by methylcholanthrene; by simian virus 40, Kirsten and Moloney murine sarcoma viruses, simian sarcoma virus, and adenovirus; and by a "spontaneous" event in culture were examined for the expression of receptors for the platelet-derived <em>growth</em> <em>factor</em> (PDGF) and for production of substances able to compete with 125I-labeled PDGF for binding to the cell-surface PDGF receptor. In each case, transformation resulted in a 50-100% decrease in available PDGF receptors. All transformed cells except the methylcholanthrene-transformed mouse cells produce a PDGF competitor into the conditioned medium. Levels of PDGF competitor in conditioned medium at the end of a 48-hr collection were as high as 2 ng/ml--high enough to be measured by radioreceptor assay diluted 1:30 and to maximally stimulate [3H]thymidine incorporation by human <em>fibroblasts</em>. The PDGF competitor activity detected in a radioreceptor assay does not reflect irreversible (e.g., proteolytic) damage to the receptor of test cells since its effects are reversed by acetic acid dissociation. Antiserum against human PDGF neutralizes <em>20</em>-80% of the PDGF competitor found in conditioned medium from different transformed human cells and 100% of the activity from normal human endothelial cells. The possibility that induction of expression of the cellular PDGF gene may be involved in the mechanism of transformation of PDGF-responsive mesenchymal cells is discussed.

Addition of platelet-derived <em>growth</em> <em>factor</em>, epidermal <em>growth</em> <em>factor</em>, or serum to quiescent human <em>fibroblasts</em>, loaded with the fluorescent Ca2+ indicator quin-2, causes an immediate, up to 3-fold, rise in cytoplasmic free Ca2+ concentration [( Ca2+]i). In contrast, insulin and tumor-promoting phorbol ester have no effect on [Ca2+]i. The mitogen-induced [Ca2+]i response is initiated within a few s, reaches a maximum by <em>20</em>-40 s, and then slowly declines to a new steady level. The [Ca2+]i response is not prevented by removal of external Ca2+ and is independent of the transmembrane Na+ gradient and membrane potential. It is concluded that platelet-derived <em>growth</em> <em>factor</em>, epidermal <em>growth</em> <em>factor</em>, and serum rapidly mobilize Ca2+ from intracellular stores, presumably due to the prior breakdown of inositol phospholipids, and that the resulting rise in [Ca2+]i may function as an initial signal in <em>growth</em> <em>factor</em> action.

Primary embryonic hippocampal neurons can develop morphologically and functionally in culture but do not survive more than a few weeks. It has been reported that basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) promotes the survival of and neurite elongation from fetal hippocampal neurons. We report that bFGF, in a dose-dependent manner, can induce the survival (50 pg to 1 ng/ml) and proliferation (10-<em>20</em> ng/ml) of embryonic hippocampal progenitor neurons in vitro. In serum-free medium containing high concentrations of bFGF, neurons not only proliferated (4-day doubling time) and differentiated morphologically but also could be passaged and grown as continuous cell lines. The neuronal nature of the proliferating cells was positively established by immunostaining with several different neuron-specific markers and by detailed ultrastructural analyses. The proliferative effect of bFGF was used to generate nearly pure neuronal cell cultures that can be passaged, frozen, thawed, and cultured again. Neurons have been maintained>> 5 months in culture. The ability to establish long-term primary neuronal cultures offers the possibility that clonal lines of distinct neuronal cell types may be isolated from specific areas of the central nervous system. Such long-term neuronal cultures should prove valuable in studying neurons at the individual cell level and also in exploring interactions between neurons in vitro. The observed dose dependence raises the possibility that cell survival and proliferation in vivo may be influenced by different levels of bFGF.