Extracellular matrix controls capillary endothelial cell sensitivity to soluble mitogens by binding integrin receptors and thereby activating a chemical signaling response that rapidly integrates with <em>growth</em> <em>factor</em>-induced signaling mechanisms. Here we report that in addition to integrins, <em>growth</em> <em>factor</em> receptors and multiple molecules that transduce signals conveyed by both types of receptors are immobilized on the cytoskeleton (CSK) and spatially integrated within the focal adhesion complex (FAC) at the site of integrin binding. FACs were rapidly induced in round cells and physically isolated from the remainder of the CSK after detergent-extraction using magnetic microbeads coated with fibronectin or a synthetic RGD-containing peptide. Immunofluorescence microscopy revealed that multiple signaling molecules (e.g., pp60c-src, pp125FAK, phosphatidylinositol-3-kinase, phospholipase C-gamma, and Na+/H+ antiporter) involved in both integrin and <em>growth</em> <em>factor</em> receptor signaling pathways became associated with the CSK framework of the FAC within <em>15</em> min after binding to beads coated with integrin ligands. Recruitment of tyrosine kinases to the FAC was also accompanied by a local increase in tyrosine phosphorylation, as indicated by enhanced phosphotyrosine staining at the site of integrin binding. In contrast, neither recruitment of signaling molecules nor increased phosphotyrosine staining was observed when cells bound to beads coated with a control ligand (acetylated low density lipoprotein) that ligates transmembrane scavenger receptors, but does not induce FAC formation. Western blot analysis confirmed that FACs isolated using RGD-beads were enriched for pp60c-src, pp125FAK, phospholipase C-gamma, and the Na+/H+ antiporter when compared with intact CSK or basal cell surface preparations that retained lipid bilayer. Isolated FACs were also greatly enriched for the high affinity <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor flg. Most importantly, isolated FACs continued to exhibit multiple chemical signaling activities in vitro, including protein tyrosine kinase activities (pp60c-src and pp125FAK) as well as the ability to undergo multiple sequential steps in the inositol lipid synthesis cascade. These data suggest that many of the chemical signaling events that are induced by integrins and <em>growth</em> <em>factor</em> receptors in capillary cells may effectively function in a "solid-state" on insoluble CSK scaffolds within the FAC and that the FAC may represent a major site for signal integration between these two regulatory pathways. Future investigations into the biochemical and biophysical basis of signal transduction may be facilitated by this method, which results in isolation of FACs that retain the CSK framework as well as multiple associated chemical signaling activities.

The <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family consists of at least seven closely related polypeptide mitogens which exert their activities by binding and activation of specific cell surface receptors. Unanswered questions have been whether there are multiple FGF receptors and what <em>factors</em> determine binding specificity and biological response. We report the complete cDNA cloning of two human genes previously designated flg and bek. These genes encode two similar but distinct cell surface receptors comprised of an extracellular domain with three immunoglobulin-like regions, a single transmembrane domain, and a cytoplasmic portion containing a tyrosine kinase domain with a typical kinase insert. The expression of these two cDNAs in transfected NIH 3T3 cells led to the biosynthesis of proteins of <em>15</em>0 kd and 135 kd for flg and bek, respectively. Direct binding experiments with radiolabeled acidic FGF (aFGF) or basic FGF (bFGF), inhibition of binding with native <em>growth</em> <em>factors</em>, and Scatchard analysis of the binding data indicated that bek and flg bind either aFGF or bFGF with dissociation constants of (2-<em>15</em>) x 10(-11) M. The high affinity binding of two distinct <em>growth</em> <em>factors</em> to each of two different receptors represents a unique double redundancy without precedence among polypeptide <em>growth</em> <em>factor</em>-receptor interactions.

To assess whether the progression of plasma cell tumors is accompanied by angiogenesis and secretion of matrix-degrading enzymes, bone marrow biopsy specimens from 20 patients with monoclonal gammopathy of undetermined significance (MGUS), 18 patients with nonactive multiple myeloma (MM), and 26 patients with active MM were evaluated for their angiogenic potential and matrix-metalloproteinase (MMP) production. A fivefold increase of the <em>factor</em> VIII+ microvessel area was measured by a planimetric method of point counting in the bone marrow of patients with active MM as compared with nonactive MM and MGUS patients (P <.01). When serum-free conditioned media (CM) of plasma cells isolated from the bone marrow of each patient were tested in vivo for their angiogenic activity in the chick embryo chorioallantoic membrane (CAM) assay, the incidence of angiogenic samples was significantly higher (P <. 01) in the active MM group (76%) compared with nonactive MM (33%) and MGUS (20%) groups. Moreover, a linear correlation (P <.01) was found between the extent of vascularization of the bone marrow of a given patient and the angiogenic activity exerted in the CAM assay by the plasma cells isolated from the same bone marrow. In vitro, a significantly higher fraction of the plasma cell CM samples from the active MM group stimulated human umbilical vein endothelial cell (HUVEC) proliferation (53%, P <.01), migration (42%, P <.05), and/or monocyte chemotaxis (38%, P <.05) when compared with nonactive MM and MGUS groups (ranging between 5% and <em>15</em>% of the samples). Also, immunoassay of plasma cell extracts showed significantly higher (P <. 01) levels of the angiogenic basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-2 in the active MM patients than in nonactive MM and MGUS patients (<em>15</em>3 +/- 59, 23 +/- 17, and 31 +/- 18 pg FGF-2/100 micrograms of protein, respectively). Accordingly, neutralizing anti-FGF-2 antibody caused a significant inhibition (ranging from 54% to 68%) of the biological activity exerted on cultured endothelial cells and in the CAM assay by plasma cell CM samples from active MM patients. Finally, in situ hybridization of bone marrow plasma cells and gelatin-zymography of their CM showed that active MM patients express significantly higher (P <.01) levels of MMP-2 mRNA and protein when compared with nonactive MM and MGUS patients, whereas MMP-9 expression was similar in all groups. Taken together, these findings indicate that the progression of plasma cell tumors is accompanied by an increase of bone marrow neovascularization. This is paralleled by an increased angiogenic and invasive potential of bone marrow plasma cells, which is dependent, at least in part, by FGF-2 and MMP-2 production. Induction of angiogenesis and secretion of MMPs by plasma cells in active disease may play a role in their medullary and extramedullary dissemination, raising the hypothesis that angiostatic/anti-MMP agents may be used for therapy of MM.

Vertebrate limb development depends on signals from the apical ectodermal ridge (AER), which rims the distal tip of the limb bud. Removal of the AER in chick results in limbs lacking distal skeletal elements. <em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) proteins can substitute for the AER (refs 4-7), suggesting that FGF signalling mediates AER activity. Of the four mouse Fgf genes (Fgf4 , Fgf8, Fgf9, Fgf17) known to display AER-specific expression domains within the limb bud (AER-Fgfs), only Fgf8 is expressed throughout the AER. Moreover, Fgf8 expression precedes that of other AER-Fgfs (refs 8-13), suggesting that Fgf8 may perform unique functions early in limb development. In mice, loss of function of Fgf4 (refs 13,14), Fgf9 (D. Ornitz, pers. comm.) or Fgf17 (ref. <em>15</em>) has no effect on limb formation. We report here that inactivating Fgf8 in early limb ectoderm causes a substantial reduction in limb-bud size, a delay in Shh expression, misregulation of Fgf4 expression, and hypoplasia or aplasia of specific skeletal elements. Our data identify Fgf8 as the only known AER-Fgf individually necessary for normal limb development, and provide insight into the function of Fgf signalling from the AER in the normal out<em>growth</em> and patterning of the limb.

The trefoil peptides, a recently recognized family of protease-resistant peptides, expressed in a regional specific pattern throughout the normal gastrointestinal tract. Although these peptides have been hypothesized to act as <em>growth</em> <em>factors</em>, their functional properties are largely unknown. Addition of recombinant trefoil peptides human spasmolytic polypeptide (HSP), rat and human intestinal trefoil <em>factor</em> (RITF and HITF) to subconfluent nontransformed rat intestinal epithelial cell lines (IEC-6 and IEC-17), human colon cancer-derived cell lines (HT-29 and CaCO2) or nontransformed <em>fibroblasts</em> (NRK and BHK) had no significant effect on proliferation. However addition of the trefoil peptides to wounded monolayers of confluent IEC-6 cells in an in vitro model of epithelial restitution resulted in a 3-6-fold increase in the rate of epithelial migration into the wound. Stimulation of restitution by the trefoil peptide HSP was enhanced in a cooperative fashion by the addition of mucin glycoproteins purified from the colon or small intestine of either rat or man, achieving up to a <em>15</em>-fold enhancement in restitution. No synergistic effect was observed by the addition of nonmucin glycoproteins. In contrast to cytokine stimulation of intestinal epithelial cell restitution which is mediated through enhanced TGF beta bioactivity, trefoil peptide, and trefoil peptide-mucin glycoprotein stimulation of restitution was not associated with alteration in concentrations of bioactive TGF-beta and was not affected by the presence of immunoneutralizing anti-TGF beta antiserum. Collectively, these findings suggest that the trefoil peptides which are secreted onto the lumenal surface of the gastrointestinal tract may act in conjunction with the mucin glycoprotein products of goblet cells to promote reestablishment of mucosal integrity after injury through mechanisms distinct from those which may act at the basolateral pole of the epithelium.

Signal transducers and activators of transcription (STATs) were originally identified as key components of signaling pathways involved in mediating responses to IFNs. Previous studies showed that the Src oncoprotein constitutively activates one STAT family member, Stat3. In this study, we investigated STAT activation in a panel of rodent <em>fibroblast</em> cell lines stably transformed by diverse viral oncoproteins. Using a temperature-sensitive mutant of v-Src, we determined that Stat3 is activated within <em>15</em> min of shift from nonpermissive to permissive temperature for cell transformation. This finding indicates that v-Src tyrosine kinase activity is required for Stat3 activation and suggests that Stat3 is proximal to signaling initiated by Src. In addition, Stat3 activation is induced by another nonreceptor tyrosine kinase, v-Fps; by polyoma virus middle T antigen, which activates Src family kinases; and by v-Sis, which acts as a ligand for the platelet-derived <em>growth</em> <em>factor</em> receptor. In contrast SV40 large T antigen, which transforms cells through different mechanisms, and the v-Ras and v-Raf oncoproteins, which lie in signaling pathways downstream of tyrosine kinases, do not activate Stat3. We did not detect significant activation of Stat1, Stat5, or Stat6 in <em>fibroblasts</em> transformed by the viral oncoproteins investigated. Moreover, Stat3 is activated in response to epidermal <em>growth</em> <em>factor</em> (EGF) but not heregulins in immortalized normal human breast epithelial cells. Because constitutive activation of c-Src and EGF receptor kinases is associated with the progression of breast cancer, we examined activation of STATs in human cell lines derived from breast carcinomas. We detected constitutive activation of Stat3 in five of nine breast carcinoma cell lines but not in normal breast epithelial cells. Furthermore, experiments with an EGF receptor-specific inhibitor indicated that the constitutive activation of Stat3 in these breast carcinoma cell lines is not necessarily dependent on signaling through the EGF receptor, although EGF stimulation further increases Stat3 activation. Taken together, our results demonstrate that selective activation of Stat3 is a common event during oncogenic transformation that directly or indirectly involves activation of specific tyrosine kinase signaling pathways.

Regulation of hepatic carbohydrate homeostasis is crucial for maintaining energy balance in the face of fluctuating nutrient availability. Here, we show that the hormone <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em>/19 (FGF<em>15</em>/19), which is released postprandially from the small intestine, inhibits hepatic gluconeogenesis, like insulin. However, unlike insulin, which peaks in serum <em>15</em> min after feeding, FGF<em>15</em>/19 expression peaks approximately 45 min later, when bile acid concentrations increase in the small intestine. FGF<em>15</em>/19 blocks the expression of genes involved in gluconeogenesis through a mechanism involving the dephosphorylation and inactivation of the transcription <em>factor</em> cAMP regulatory element-binding protein (CREB). This in turn blunts expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and other genes involved in hepatic metabolism. Overexpression of PGC-1α blocks the inhibitory effect of FGF<em>15</em>/19 on gluconeogenic gene expression. These results demonstrate that FGF<em>15</em>/19 works subsequent to insulin as a postprandial regulator of hepatic carbohydrate homeostasis.

This study was designed to investigate the effects of intravenous administration of marrow stromal cells (MSCs) on the expression of <em>growth</em> <em>factors</em> in rat brain after traumatic brain injury (TBI). The fate of transplanted MSCs and expression of <em>growth</em> <em>factors</em> was examined by immunohistochemistry. In addition, the level of <em>growth</em> <em>factors</em> was measured quantitatively using enzyme linked immunosorbent assay (ELISA). <em>Growth</em> <em>factors</em> that were studied included nerve <em>growth</em> <em>factor</em> (NGF), brain-derived neurotrophic <em>factor</em> (BDNF), and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). For immunohistochemical studies, 12 male Wistar rats were subjected to TBI and then divided into three groups with the first group receiving no treatment, the second group receiving saline (placebo) and the third group receiving MSCs intravenously 1 day after TBI. The neurological function of rats was studied by using Rotarod motor test and modified neurological severity scores. The animals were sacrificed <em>15</em> days after TBI and brain sections stained by immunohistochemistry to study the distribution of MSCs as well as expression of <em>growth</em> <em>factors</em> NGF, BDNF, and bFGF. For quantitative analysis, a second set of male Wistar rats (n = 18) was subjected to TBI and then injected with either saline (n = 9) or MSCs (n = 9) 1 day after injury. These rats were sacrificed on days 2, 5, and 8 after TBI and brain extracts used to measure NGF, BDNF, and bFGF. We found that after transplantation, MSCs preferentially migrated into the injured hemisphere and there was a statistically significant improvement in the functional outcome of MSC-treated rats compared to control rats. NGF, BDNF, and bFGF were expressed in the injured brain of both treated as well as control rats; however, quantitative ELISA studies showed that expression of NGF and BDNF was significantly increased (p < 0.05) in the treated group. This study shows that intravenous administration of MSCs after TBI increases the expression of <em>growth</em> <em>factors</em> (NGF, BDNF), which possibly contributes to the improvement in functional outcome seen in these rats.

Activation of farnesoid X receptor (Fxr, Nr1h4) is a major mechanism in suppressing bile-acid synthesis by reducing the expression levels of genes encoding key bile-acid synthetic enzymes (e.g., cytochrome P450 [CYP]7A1/Cyp7a1 and CYP8B1/Cyp8b1). FXR-mediated induction of hepatic small heterodimer partner (SHP/Shp, Nr0b2) and intestinal <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em> (Fgf<em>15</em>; FGF19 in humans) has been shown to be responsible for this suppression. However, the exact contribution of Shp/Fgf<em>15</em> to this suppression, and the associated cell-signaling pathway, is unclear. By using novel genetically modified mice, the current study showed that the intestinal Fxr/Fgf<em>15</em> pathway was critical for suppressing both Cyp7a1 and Cyp8b1 gene expression, but the liver Fxr/Shp pathway was important for suppressing Cyp8b1 gene expression and had a minor role in suppressing Cyp7a1 gene expression. Furthermore, in vivo administration of Fgf<em>15</em> protein to mice led to a strong activation of extracellular signal-related kinase (ERK) and, to a smaller degree, Jun N-terminal kinase (JNK) in the liver. In addition, deficiency of either the ERK or JNK pathway in mouse livers reduced the basal, but not the Fgf<em>15</em>-mediated, suppression of Cyp7a1 and Cyp8b1 gene expression. However, deficiency of both ERK and JNK pathways prevented Fgf<em>15</em>-mediated suppression of Cyp7a1 and Cyp8b1 gene expression.

CONCLUSIONS

The current study clearly elucidates the underlying molecular mechanism of hepatic versus intestinal Fxr in regulating the expression of genes critical for bile-acid synthesis and hydrophobicity in the liver.

The human <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (hFGF23) and its autosomal dominant hypophosphatemic rickets (ADHR) mutant genes were incorporated into animals by naked DNA injection to investigate the action on phosphate homeostasis in vivo. The hFGF23 mutants (R176Q, R179Q, and R179W) markedly reduced serum phosphorus (6.2-6.9 mg/dl) compared with the plasmid MOCK (8.5 mg/dl). However, native hFGF23 did not affect serum phosphorus (8.6 mg/dl). Both hFGF23 and hFGF23R179Q mRNAs were expressed more than 100-fold in the liver 4 days after injection, however, the C-terminal portion of hFGF23 was detected only in the serum from hFGF23R179Q-injected animals (1109 pg/ml). hFGF23R179Q mutant was secreted as a 32-kDa protein, whereas, native hFGF23 was detected as a 20-kDa protein in the cell-conditioned media. These results suggest the hFGF23R179Q protein is resistant to intracellular proteolytic processing. The hFGF23R179Q suppressed Na/P(i) co-transport activities both in kidney and in small intestine by 45 and 30%, respectively, as well as serum 1alpha,25-dihydroxyvitamin D(3) to less than <em>15</em> pg/ml. However, it had little effect on serum parathyroid hormone (PTH). Infusion of hFGF23R179Q protein normalized serum phosphorus in thyroparathyroidectomized rats without affecting serum calcium. Taken together, the FGF23 mutants reduce both phosphate uptake in intestine and phosphate reabsorption in kidney, independent of PTH action.

OBJECTIVE

The present study was designed to decipher molecular mechanisms underlying nicotine's promoting atrial fibrillation (AF) by inducing atrial structural remodelling.

RESULTS

The canine model of AF was successfully established by nicotine administration and rapid pacing. The atrial <em>fibroblasts</em> isolated from healthy dogs were treated with nicotine. The role of microRNAs (miRNAs) on the expression and regulation of transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1), TGF-beta receptor type II (TGF-betaRII), and collagen production was evaluated in vivo and in vitro. Administration of nicotine for 30 days increased AF vulnerability by approximately eight- to <em>15</em>-fold in dogs. Nicotine stimulated remarkable collagen production and atrial fibrosis both in vitro in cultured canine atrial <em>fibroblasts</em> and in vivo in atrial tissues. Nicotine produced significant upregulation of expression of TGF-beta1 and TGF-betaRII at the protein level, and a 60-70% decrease in the levels of miRNAs miR-133 and miR-590. This downregulation of miR-133 and miR-590 partly accounts for the upregulation of TGF-beta1 and TGF-betaRII, because our data established TGF-beta1 and TGF-betaRII as targets for miR-133 and miR-590 repression. Transfection of miR-133 or miR-590 into cultured atrial <em>fibroblasts</em> decreased TGF-beta1 and TGF-betaRII levels and collagen content. These effects were abolished by the antisense oligonucleotides against miR-133 or miR-590. The effects of nicotine were prevented by an alpha7 nicotinic acetylcholine receptor antagonist.

CONCLUSIONS

We conclude that the profibrotic response to nicotine in canine atrium is critically dependent upon downregulation of miR-133 and miR-590.

Formal proof for an involvement of autocrine stimulation in the disturbed <em>growth</em> of malignant cells has been difficult to obtain, in part due to lack of precise methods of assessing <em>growth</em> <em>factor</em> production and receptor occurrence. In this study we have analyzed the mRNA levels for two <em>growth</em> <em>factors</em> and the corresponding receptors in a number of established human malignant glioma cell lines. Twenty-one tested lines all contained transcripts for the platelet-derived <em>growth</em> <em>factor</em> (PDGF) A chain while 16-17 of 21 expressed the c-sis/PDGF B chain gene; these two genes were expressed independently of each other. PDGF receptor transcripts were present in <em>15</em>-16 of the 21 lines. Transcripts for the epidermal <em>growth</em> <em>factor</em> receptor were found in all <em>15</em> tested lines, in 2 of them at high levels, and the corresponding ligand transforming <em>growth</em> <em>factor</em>-alpha was found in 11 of <em>15</em> lines. No amplification or structural rearrangements of the genes, as analyzed by Southern blot hybridization, could explain the varying expression of PDGF A and B chain transcripts or the elevated levels of epidermal <em>growth</em> <em>factor</em> receptor mRNA. A correlation was found between cell morphology and expression of <em>growth</em> <em>factor</em> and receptor mRNA in these lines. The highest amount of PDGF receptor transcripts was found in cells with <em>fibroblast</em>-like morphology, and c-sis/B chain transcripts were found in small cell types and in cells with astrocyte-like morphology, while no clear relationship was found between PDGF receptor and A chain transcript levels or between morphology and A chain transcripts. It is possible that the findings reflect a coordinated expression of these genes in the progenitor cells. In conclusion, the data imply the existence of two possible autocrine loops in human malignant glioma lines, affecting the PDGF and epidermal <em>growth</em> <em>factor</em> receptor pathways.

BACKGROUND

The c-Myc transcription factor is a master regulator and integrates cell proliferation, cell growth and metabolism through activating thousands of target genes. Our identification of direct c-Myc target genes by chromatin immunoprecipitation (ChIP) coupled with pair-end ditag sequencing analysis (ChIP-PET) revealed that nucleotide metabolic genes are enriched among c-Myc targets, but the role of Myc in regulating nucleotide metabolic genes has not been comprehensively delineated.

RESULTS

Here, we report that the majority of genes in human purine and pyrimidine biosynthesis pathway were induced and directly bound by c-Myc in the P493-6 human Burkitt's lymphoma model cell line. The majority of these genes were also responsive to the ligand-activated Myc-estrogen receptor fusion protein, Myc-ER, in a Myc null rat fibroblast cell line, HO.15 MYC-ER. Furthermore, these targets are also responsive to Myc activation in transgenic mouse livers in vivo. To determine the functional significance of c-Myc regulation of nucleotide metabolism, we sought to determine the effect of loss of function of direct Myc targets inosine monophosphate dehydrogenases (IMPDH1 and IMPDH2) on c-Myc-induced cell growth and proliferation. In this regard, we used a specific IMPDH inhibitor mycophenolic acid (MPA) and found that MPA dramatically inhibits c-Myc-induced P493-6 cell proliferation through S-phase arrest and apoptosis.

CONCLUSIONS

Taken together, these results demonstrate the direct induction of nucleotide metabolic genes by c-Myc in multiple systems. Our finding of an S-phase arrest in cells with diminished IMPDH activity suggests that nucleotide pool balance is essential for c-Myc's orchestration of DNA replication, such that uncoupling of these two processes create DNA replication stress and apoptosis.

Bidirectional communication between oocytes and the companion granulosa cells is essential for the development and functions of both compartments. Oocytes are deficient in their ability to transport certain amino acids and in carrying out glycolysis and cholesterol biosynthesis. Cumulus cells must provide them with the specific amino acids and the products in these metabolic pathways. Oocytes control metabolic activities in cumulus cells by promoting the expression of genes in cumulus cells encoding specific amino acid transporters and enzymes essential for the oocyte-deficient metabolic processes. Hence oocytes outsource metabolic functions to cumulus cells to compensate for oocyte metabolic deficiencies. Oocyte control of granulosa cell metabolism may also participate in regulating the rate of follicular development in coordination with endocrine, paracrine, and autocrine signals. Oocytes influence granulosa cell development mainly by secretion of paracrine <em>factors</em>, although juxtacrine signals probably also participate. Key oocyte-derived paracrine <em>factors</em> include <em>growth</em> differentiation <em>factor</em> 9, bone morphogenetic protein <em>15</em>, and <em>fibroblast</em> <em>growth</em> <em>factor</em> 8B.

BACKGROUND

Recent studies have suggested that vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) may have synergistic effects on the induction of angiogenesis in vitro. Therefore, we investigated the hypothesis that the simultaneous administration of VEGF and bFGF, each having been previously shown to independently enhance collateral development in an animal model of hind limb ischemia, could have a synergistic effect in vivo.

RESULTS

Ten days after surgical induction of unilateral hind limb ischemia, New Zealand White rabbits were randomized to receive either VEGF 500 micrograms alone (n = 6), bFGF 10 micrograms alone (n = 7), VEGF 500 micrograms, immediately followed by 10 micrograms bFGF (n = 7), or vehicle only (control animals, n = 8) in each case administered intra-arterially via a catheter in the internal iliac artery of the ischemic limb. BP ratio (BPR, ischemic/healthy limb) at day 10 for the VEGF+bFGF group was 0.82 +/- 0.01, much superior (P < .0005) to that of either the VEGF group (0.52 +/- 0.02) or the bFGF group (0.57 +/- 0.02). This outcome persisted at day 30: BPR in the VEGF+bFGF group (0.91 +/- 0.02) exceeded that of the control group (0.49 +/- 0.05, P < .0001), the VEGF group (0.65 +/- 0.03, P < .0005), or the bFGF group (0.66 +/- 0.03, P < .0005). Serial angiography demonstrated a progressive increase in luminal diameter of the stem collateral artery and the number of opacified collaterals in the thigh of the ischemic limbs in all groups. Stem artery diameter with VEGF+bFGF (1.34 +/- 0.07 mm) on day 30 was significantly (P < .05) greater than with either VEGF (1.09 +/- 0.09) or bFGF (1.18 +/- 0.06) alone. Capillary density was significantly greater (P < .05) in VEGF+bFGF animals (275 +/- 20 mm2) compared with VEGF (201 +/- 8) or bFGF (209 +/- 15).

CONCLUSIONS

Combined administration of VEGF and bFGF stimulates significantly greater and more rapid augmentation of collateral circulation, resulting in superior hemodynamic improvement compared with either VEGF or bFGF alone. This synergism of two angiogenic mitogens with different target cell specificities may have important implications for the treatment of severe arterial insufficiency in patients whose disease is not amenable to direct revascularization.

We measured the content and metabolism of histone mRNA in mouse 3T6 <em>fibroblasts</em> during a serum-induced transition from the resting to <em>growing</em> state. The content of several histone H3 and H2b mRNAs was measured by an S1 nuclease procedure. All of these increase in parallel by a <em>factor</em> of about 50 during S phase. However, the rate of H3 gene transcription increased only fivefold during this period, as determined in an in vitro transcription assay. This suggests that histone mRNA content is also controlled at the posttranscriptional level. When resting cells were serum stimulated in the presence of cytosine arabinoside, the rate of H3 gene transcription increased to about the same extent as that in control-stimulated cells. However, cytoplasmic H3 mRNA content increased only five to seven-fold. The half-life of H3 mRNA during S phase was about 4 to 5 h. When cytosine arabinoside was added to cells in the S phase, the half-life of the message decreased to about <em>15</em> min. The rapid turnover of H3 mRNA was prevented when the drug was added in the presence of cycloheximide or puromycin. The rate of H3 gene transcription decreased by only 35% after treatment with cytosine arabinoside. These results suggest that H3 gene transcription is not tightly coupled to DNA replication but is controlled temporally during the resting to <em>growing</em> transition. However, there is a correlation between the rate of DNA synthesis and the stability of histone H3 mRNA.

Fish is a scaffolding protein and Src substrate. It contains an amino-terminal Phox homology (PX) domain and five Src homology 3 (SH3) domains, as well as multiple motifs for binding both SH2 and SH3 domain-containing proteins. We have determined that the PX domain of Fish binds 3-phosphorylated phosphatidylinositols (including phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate). Consistent with this, a fusion protein of green fluorescent protein and the Fish PX domain localized to punctate structures similar to endosomes in normal <em>fibroblasts</em>. However, the full-length Fish protein was largely cytoplasmic, suggesting that its PX domain may not be able to make intermolecular interactions in unstimulated cells. In Src-transformed cells, we observed a dramatic re-localization of some Fish molecules to actin-rich structures called podosomes; the PX domain was both necessary and sufficient to effect this translocation. We used a phage display screen with the fifth SH3 domain of Fish and isolated ADAM19 as a binding partner. Subsequent analyses in mammalian cells demonstrated that Fish interacts with several members of the ADAMs family, including ADAMs 12, <em>15</em>, and 19. In Src-transformed cells, ADAM12 co-localized with Fish in podosomes. Because members of the ADAMs family have been implicated in <em>growth</em> <em>factor</em> processing, as well as cell adhesion and motility, Fish could be acting as an adaptor molecule that allows Src to impinge on these processes.

Rheumatoid arthritis (RA) is accompanied by synovial inflammation, proliferation, and cartilage destruction. The reasons the activation of synovial <em>fibroblasts</em> often persists despite antiinflammatory therapy are not known. One possibility is that the synovial membrane becomes gradually repopulated with immature mesenchymal and bone marrow cells with altered properties. To explore this hypothesis, we have investigated the expression in RA synovial tissues of various embryonic <em>growth</em> <em>factors</em> from the wingless (wnt) and frizzled (fz) families, which have been implicated in cell-fate determination in both bone marrow progenitors and limb-bud mesenchyme. Reverse transcriptase-PCR analysis revealed expression of five wnt (wnt1, 5a, 10b, 11, and 13) and three fz (fz2, 5, and 7) isoforms in RA synovial tissues. Osteoarthritis synovial tissues expressed much less wnt5a and fz5. Northern blotting confirmed the overexpression of wnt5a and fz5 in RA synovial tissues, in comparison to a panel of normal adult tissues. Compared with normal synovial <em>fibroblasts</em>, cultured RA <em>fibroblast</em>-like synoviocytes expressed higher levels of IL-6, IL-8, and IL-<em>15</em>. Transfection of normal <em>fibroblasts</em> with a wnt5a expression vector reproduced this pattern of cytokine expression and stimulated IL-<em>15</em> secretion. These results suggest that the unusual phenotypic properties of RA <em>fibroblasts</em> may be attributable partly to their replacement with primitive <em>fibroblast</em>-like synoviocytes with characteristics of immature bone marrow and mesenchymal cells. Clear delineation of the signaling pathway(s) initiated by the wnt5a/fz5 ligand-receptor pair in the RA synovium may yield new targets for therapeutic intervention.

Injury to the central nervous system (CNS) by ionizing radiation may be a consequence of damage to the vascular endothelium. Recent studies showed that radiation-induced apoptosis of endothelial cells in vitro and in the lung in vivo is mediated by the lipid second messenger ceramide via activation of acid sphingomyelinase (ASM). This apoptotic response to radiation can be inhibited by basic <em>fibroblast</em> <em>growth</em> <em>factor</em> or by genetic mutation of ASM. In the CNS, single-dose radiation has been shown to result in a <em>15</em>% loss of endothelial cells within 24 h, but whether or not this loss is associated with apoptosis remains unknown. In the present studies, dose- and time-dependent induction of apoptosis was observed in the C57BL/6 mouse CNS. Apoptosis was quantified by terminal deoxynucleotidyl transferase-mediated nick end labeling, and specific endothelial apoptosis was determined by histochemical double labeling with terminal deoxynucleotidyl transferase-mediated nick end labeling and Lycopersicon esculentum lectin. Beginning at 4 h after single-dose radiation, apoptosis was ongoing for 24 h and peaked at 12 h at an incidence of 0.7-1.4% of the total cells in spinal cord sections. Up to 20% of the apoptotic cells were endothelial. This effect was also seen in multiple regions of the brain (medulla, pons, and hippocampus). A significant reduction of radiation-induced apoptosis was observed after i.v. basic <em>fibroblast</em> <em>growth</em> <em>factor</em> treatment (0.45-4.5 microg/mouse). Identical results were noted in C3H/HeJ mice. Furthermore, irradiated ASM knockout mice displayed as much as a 70% reduction in endothelial apoptosis. This study demonstrates that ionizing radiation induces early endothelial cell apoptosis throughout the CNS. These data are consistent with recent evidence linking radiation-induced stress with ceramide and suggest approaches to modify the apoptotic response in control of radiation toxicity in the CNS.

Sphingolipids (SLs) are plasma membrane constituents in eukaryotic cells which play important roles in a wide variety of cellular functions. However, little is known about the mechanisms of their internalization from the plasma membrane or subsequent intracellular targeting. We have begun to study these issues in human skin <em>fibroblasts</em> using fluorescent SL analogues. Using selective endocytic inhibitors and dominant negative constructs of dynamin and epidermal <em>growth</em> <em>factor</em> receptor pathway substrate clone <em>15</em>, we found that analogues of lactosylceramide and globoside were internalized almost exclusively by a clathrin-independent ("caveolar-like") mechanism, whereas an analogue of sphingomyelin was taken up approximately equally by clathrin-dependent and -independent pathways. We also showed that the Golgi targeting of SL analogues internalized via the caveolar-like pathway was selectively perturbed by elevated intracellular cholesterol, demonstrating the existence of two discrete Golgi targeting pathways. Studies using SL-binding toxins internalized via clathrin-dependent or -independent mechanisms confirmed that endogenous SLs follow the same two pathways. These findings (a) provide a direct demonstration of differential SLs sorting into early endosomes in living cells, (b) provide a "vital marker" for endosomes derived from caveolar-like endocytosis, and (c) identify two independent pathways for lipid transport from the plasma membrane to the Golgi apparatus in human skin <em>fibroblasts</em>.

The <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family consists of at least <em>15</em> structurally related polypeptide <em>growth</em> <em>factors</em>. Their expression is controlled at the levels of transcription, mRNA stability, and translation. The bioavailability of FGFs is further modulated by posttranslational processing and regulated protein trafficking. FGFs bind to receptor tyrosine kinases (FGFRs), heparan sulfate proteoglycans (HSPG), and a cysteine-rich FGF receptor (CFR). FGFRs are required for most biological activities of FGFs. HSPGs alter FGF-FGFR interactions and CFR participates in FGF intracellular transport. FGF signaling pathways are intricate and are intertwined with insulin-like <em>growth</em> <em>factor</em>, transforming <em>growth</em> <em>factor</em>-beta, bone morphogenetic protein, and vertebrate homologs of Drosophila wingless activated pathways. FGFs are major regulators of embryonic development: They influence the formation of the primary body axis, neural axis, limbs, and other structures. The activities of FGFs depend on their coordination of fundamental cellular functions, such as survival, replication, differentiation, adhesion, and motility, through effects on gene expression and the cytoskeleton.

Transforming <em>growth</em> <em>factor</em>-alpha (TGFalpha) and <em>fibroblast</em> <em>growth</em> <em>factor</em>-7 (FGF7) exhibit distinct expression patterns in the mammary gland. Both <em>factors</em> signal through mitogen-activated kinase/extracellular regulated kinase-1,2 (MAPK(ERK1,2)); however, their unique and/or combined contributions to mammary morphogenesis have not been examined. In ex vivo mammary explants, we show that a sustained activation of MAPK(ERK1,2) for 1 h, induced by TGFalpha, was necessary and sufficient to initiate branching morphogenesis, whereas a transient activation (<em>15</em> min) of MAPK(ERK1,2), induced by FGF7, led to <em>growth</em> without branching. Unlike TGFalpha, FGF7 promoted sustained proliferation as well as ectopic localization of, and increase in, keratin-6 expressing cells. The response of the explants to FGF10 was similar to that to FGF7. Simultaneous stimulation by FGF7 and TGFalpha indicated that the FGF7-induced MAPK(ERK1,2) signaling and associated phenotypes were dominant: FGF7 may prevent branching by suppression of two necessary TGFalpha-induced morphogenetic effectors, matrix metalloproteinase-3 (MMP-3/stromelysin-1), and fibronectin. Our findings indicate that expression of morphogenetic effectors, proliferation, and cell-type decisions during mammary organoid morphogenesis are intimately dependent on the duration of activation of MAPK(ERK1,2) activation.

In vitro Kaposi's sarcoma-associated herpesvirus (KSHV) infection of primary human dermal microvascular endothelial (HMVEC-d) cells and human foreskin <em>fibroblast</em> (HFF) cells is characterized by the induction of preexisting host signal cascades, sustained expression of latency-associated genes, transient expression of a limited number of lytic genes, and induction of several cytokines, <em>growth</em> <em>factors</em>, and angiogenic <em>factors</em>. Since NF-kappaB is a key molecule involved in the regulation of several of these <em>factors</em>, here, we examined NF-kappaB induction during de novo infection of HMVEC-d and HFF cells. Activation of NF-kappaB was observed as early as 5 to <em>15</em> min postinfection by KSHV, and translocation of p65-NF-kappaB into nuclei was detected by immunofluorescence assay, electrophoretic mobility shift assay, and p65 enzyme-linked immunosorbent assay. IkappaB phosphorylation inhibitor (Bay11-7082) reduced this activation significantly. A sustained moderate level of NF-kappaB induction was seen during the observed 72 h of in vitro KSHV latency. In contrast, high levels of ERK1/2 activation at earlier time points and a moderate level of activation at later times were observed. p38 mitogen-activated protein kinase was activated only at later time points, and AKT was activated in a cyclic manner. Studies with UV-inactivated KSHV suggested a role for virus entry stages in NF-kappaB induction and a requirement for KSHV viral gene expression in sustained induction. Inhibition of NF-kappaB did not affect target cell entry by KSHV but significantly reduced the expression of viral latent open reading frame 73 and lytic genes. KSHV infection induced the activation of several host transcription <em>factors</em>, including AP-1 family members, as well as several cytokines, <em>growth</em> <em>factors</em>, and angiogenic <em>factors</em>, which were significantly affected by NF-kappaB inhibition. These results suggest that during de novo infection, KSHV induces sustained levels of NF-kappaB to regulate viral and host cell genes and thus possibly regulates the establishment of latent infection.

Medium conditioned by human embryo <em>fibroblasts</em> breaks structural junctions between several types of epithelial cells, leading to separation and scattering of the cells. An assay developed in MDCK cells shows activity up to a dilution of at least 1 in 64, equivalent to less than 100 ng of total protein. The activity is non-dialysable, heat-labile, and sensitive to trypsin, and it is assumed to be due to one or more proteins. After addition of the <em>factor</em>, separation of MDCK cells begins in about <em>15</em> min and is complete in 10 h. It increases migration of MDCK cells into wounds, and causes collapse of domes. Locomotion of isolated cells is not enhanced, but cell shape is affected by local membrane movement. Under the culture conditions used the <em>factor</em>, or an associated protein, causes a weak inhibition of cell <em>growth</em> without cytotoxic activity. The scattering <em>factor</em> has not been purified, nor has a physiological role been identified, but it might be concerned in the mobilization of epithelial cells.