The increase in the amount of airway smooth muscle in the bronchial wall associated with asthma is partly due to hyperplasia. It is therefore important to determine which <em>factors</em> regulate <em>growth</em> and especially proliferation. In this study, we describe the effect of interleukin-4 (IL-4), a mast cell- and T lymphocyte-derived cytokine, on human airway smooth muscle proliferation as determined by [3H]thymidine uptake in the presence of fetal bovine serum (FBS), platelet-derived <em>growth</em> <em>factor</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and thrombin. IL-4 (5, 15, 50, and 150 ng/ml) significantly decreased 10% FBS-induced proliferation by 50, 73, 43, and 46%, respectively. The proliferative responses to platelet-derived <em>growth</em> <em>factor</em> (<em>20</em> and 40 ng/ml), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (30 ng/ml), and thrombin (1 and 10 U/ml) were significantly reduced by 19, 21, 37, 36, and 57% respectively in the presence of 50 ng/ml of IL-4. We investigated the effect of IL-4 and other known inhibitors of smooth muscle proliferation, namely PGE2, heparin, and forskolin, on intracellular cAMP concentrations. IL-4 (50 ng/ml) and heparin (100 U/ml) did not alter intracellular cAMP levels when cells were treated with 1 or 10% FBS. PGE2 (1 microM) and forskolin (10 microM) significantly increased cAMP concentration above the control value in nonproliferating cells (1% FBS treated) by 7- and 37-fold, respectively. The effect of IL-4 (50 ng/ml), PGE2 (1 microM), and forskolin (10 microM) on cyclin D1 protein expression in 10% FBS-stimulated human airway smooth muscle cells was also examined. PGE2 and forskolin did not significantly inhibit cyclin D1 expression. However, IL-4 decreased cyclin D1 expression by 21%. These results provide evidence that IL-4 decreases human airway smooth muscle cell proliferation via a mechanism that is cAMP independent and mediated, in part, by a decrease in cyclin D1 protein expression.

BACKGROUND

Approximately <em>20</em>0,000 incisional hernias are repaired annually in the United States. The high incidence (11-<em>20</em>%) and recurrence rate (24-54%) for incisional hernias have not changed appreciably in 75 years. Mechanical advances in suture material, incision orientation, and closure technique have failed to eliminate this common surgical complication. A biological approach to acute wound failure may offer a new strategy.

METHODS

A rodent incisional hernia model was used. Seventy rats underwent 5-cm midline celiotomies and were closed with fine, fast-absorbing sutures to induce intentional acute wound failure. Group 1 received no other treatment. The midline fascia in groups 2 and 3 was injected immediately prior to incision with 100 microl of vehicle alone or vehicle containing 1 microg of transforming growth factor beta(2) (TGF-beta(2)). Necropsy was performed on Postoperative Day 28 and the wounds were examined for herniation.

RESULTS

Incisional hernias developed in 88% (35/40) and 79% (11/14) of untreated incisions and those treated with vehicle alone. No hernias formed in the TGF-beta(2)-treated incisions (0/16, P < 0.05). Standard histology and immunohistochemistry demonstrated enhanced macrophage, lymphocyte, and fibroblast chemotaxis and increased collagen I and III production in TGF-beta(2) treated incisions.

CONCLUSIONS

Treatment of abdominal wall fascial incisions with TGF-beta(2) prevented the development of incisional hernias in this rat model. TGF-beta(2) stimulated fascial macrophage and fibroblast chemotaxis as well as acute wound collagen production. A biological approach such as this may reduce the incidence of incisional hernia formation in humans.

A close relationship between tumor angiogenesis, <em>growth</em>, and carcinomatosis has been observed. Netrin-4 (NT-4) has been shown to regulate angiogenic responses. We aimed to examine the effects of NT-4 on colon tumor angiogenesis, <em>growth</em>, and carcinomatosis. We showed that NT-4 was expressed in human colon cancer cells (LS174). A <em>20</em>-fold increase in NT-4 expression was stably induced by NT-4 pcDNA in LS174 cells. In vivo, a Matrigel angiogenesis assay showed that NT-4 overexpression altered vascular endothelial <em>growth</em> <em>factor</em> (VEGF)/basic <em>fibroblast</em> <em>growth</em> <em>factor</em>-induced angiogenesis. In nude mice with LS174 xenografts, NT-4 overexpression inhibited tumor angiogenesis and <em>growth</em>. In addition, these NT-4-involved inhibitory effects were associated with decreased tumor cell proliferation and increased tumor cell apoptosis. Using an orthotopic peritoneal carcinomatosis model, we demonstrated that NT-4 overexpression decreased colorectal cancer carcinomatosis. Moreover, carcinomatosis-related ascites formation was significantly decreased in mice transplanted with NT-4 LS174 cells versus control LS174 cells. The antiangiogenic activity of NT-4 was probably mediated by binding to its receptor neogenin. Netrin-4 had a direct effect on neither in vitro apoptosis and proliferation of cultured LS174 cells nor the VEGF-induced acute increase in vascular permeability in vivo. We propose that NT-4 overexpression decreases tumor <em>growth</em> and carcinomatosis, probably via an antiangiogenic effect, underlying the potential therapeutic interest in NT-4 in the treatment of colorectal cancer <em>growth</em> and carcinomatosis.

OBJECTIVE

Fibroblast growth factor 23 (FGF23) is a phosphaturic factor that is elevated in several diseases associated with hypophosphatemia and rickets. Rickets in the absence of vitamin D deficiency has been reported in African and Asian populations with a low calcium intake but the definition of risk factors has proved elusive. The aim of the study was to characterize the biochemical profile and measure FGF23 in a series of Gambian children who had presented with rickets of unknown etiology and a plasma 25-hydroxyvitamin D (25OHD) above the range typical of vitamin D-deficiency rickets.

METHODS

The 46 patients (30 males, 16 females) had bone deformities typical of rickets and were 1.1-16.4 years old (geometric mean, 3.4 years). Active rickets (on radiographs and/or elevated plasma alkaline phosphatase) was present in 28%. Plasma 25-hydroxyvitamin D was above 20 nmol/l in all patients. Concentrations of plasma FGF23, phosphate and other relevant biochemical analytes were measured in stored samples of fasting, early morning plasma and compared with those measured in samples collected from local children and stored under similar conditions.

RESULTS

The rickets patients had lower plasma phosphate, lower 25-hydroxyvitamin D, higher 1,25-dihydroxyvitamin D and elevated total alkaline phosphatase than local children. Those with active rickets had raised parathyroid hormone concentration. The patients had significantly higher FGF23 concentration than local children (geometric mean (-1SD, +1SD, range) RU/ml: 367 (87, 1552, 46-7052, n=39) vs 51 (23, 112, 3-130, n=30), p<or=0.001). At presentation, the majority (74%) had an FGF23 concentration that was above the range seen in local children, some grossly so (up to 50-fold). There was no significant difference in FGF23 concentration between those with active rickets and the other patients. Plasma phosphate was significantly and inversely correlated with FGF23 concentration. Some clinical improvements were noted after 6-12 months, during which time calcium and vitamin D had been prescribed, but FGF23 remained elevated in many patients.

CONCLUSIONS

These data suggest that perturbations of phosphate and FGF23 regulation may be implicated in the pathogenesis of calcium-deficiency rickets in Africa and Asia.

This protocol has been designed to generate neural precursor cells (NPCs) from human embryonic stem cells (hESCs) using a physiological oxygen (O(2)) level of 3% (previously termed hypoxia) and chemically defined conditions. The first stage involves suspension culture of hESC colonies at 3% O(2), where they acquire a neuroepithelial identity over a period of 2 weeks. This timescale is comparable to that observed at <em>20</em>% O(2), but survival is enhanced. Sequential application of retinoic acid and purmorphamine (PM), from day 14 to day 28, directs differentiation toward spinal motor neurons. Alternatively, addition of <em>fibroblast</em> <em>growth</em> <em>factor</em>-8 and PM generates midbrain dopaminergic neurons. OLIG2 (encoding oligodendrocyte lineage transcription <em>factor</em> 2) induction in motor neuron precursors is twofold greater than that at <em>20</em>% O(2), whereas EN1 (encoding engrailed homeobox 1) expression is enhanced fivefold. NPCs (at 3% O(2)) can be differentiated into all three neural lineages, and such cultures can be maintained long term in the absence of neurotrophins. The ability to generate defined cell types at 3% O(2) should represent a significant advancement for in vitro disease modeling and potentially for cell-based therapies.

Hepatoma-derived <em>growth</em> <em>factor</em> (HDGF), unrelated to hepatocyte <em>growth</em> <em>factor</em>, is a heparin-binding protein originally purified from human hepatoma HuH-7 cells. HDGF exhibits mitogenic activities for certain hepatoma cells, <em>fibroblasts</em> and vascular smooth muscle cells, and angiogenic activities through nuclear targeting. Recently, HDGF was found to be a mitogen for lung epithelial cells in vitro and in vivo. This suggests that HDGF may play a critical role in the development and progression of lung cancer. We investigated, immunohistochemically, the relationship between HDGF expression and clinicopathological variables, and the prognostic significance of HDGF in 102 patients with completely resected non-small-cell lung cancer (NSCLC: 70 adenocarcinomas and 32 squamous cell carcinomas). To address the mechanism of action of HDGF, we evaluated the contribution of HDGF to tumor cell proliferation and intratumor angiogenesis using anti-Ki-67 and anti-CD31 antibodies, respectively. HDGF expression was strongly detected in the nucleus of cancer cells; the HDGF-labeling index (LI) was <em>20</em>-95% (median 64.5%). There was no significant association between HDGF-expression level and clinicopathological variables. Patients with NSCLC showing a high HDGF-LI >> or =65%) had significantly worse overall and disease-free survivals than those with NSCLC showing a low HDGF-LI. Multivariate analysis revealed that HDGF is a significant independent prognostic <em>factor</em>, more powerful than pathological stage. Moreover, HDGF expression correlated with Ki-67-LI and intratumor microvessel density. We consider HDGF as a useful prognostic marker for patients with completely resected NSCLC and it may play a critical role in the pathobiology of lung cancer through its mitogenic and angiogenic activities.

Accumulating data suggest that <em>20</em>% O(2) causes human and mouse placental trophoblast stem cell (TSC) differentiation and suppresses proliferation. We tested the hypotheses that phosphorylated stress-activated protein kinase (pSAPK) levels report the optimal O(2) level for TSC culture, and that pSAPK responds to contradictory signals. We tested the dose range of 0-<em>20</em>% O(2) (0, 0.5, 2, and <em>20</em>%) on five effects in cultured TSC. The results showed 1) TSC accumulation rates were highest at 2% O(2), lower at <em>20</em>% and lowest at 0-0.5%; 2) pSAPK protein levels were lowest at 2% O(2), higher at <em>20</em>%, and highest at 0-0.5%; 3) Cleaved caspase 3, an apoptosis marker, increased at 0.5% O(2), and was highest at 0% O(2); 4) Three markers for multipotency were highest at 2 and <em>20</em>% and significantly decreased at 0.5%-0%; 5) In contrast three differentiation markers were lowest at 2% and highest at 0.5%-0%. Thus, 2% O(2) is the optimum as defined by lowest pSAPK and differentiation markers and highest <em>growth</em> rate and multipotency markers, without appreciable apoptosis. In addition, two lines of evidence suggest that <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)4 does not directly activate SAPK. SAPK activity increases transiently with FGF4 removal at 2% O(2), but SAPK activity decreases when O(2) is switched from <em>20</em>% to 2% with FGF4 present. Thus, SAPK is activated by contradictory signals, but activity decreases when either signal is removed. Taken together, the findings suggest that pSAPK senses suboptimal signals during TSC culture and probably in vivo.

OBJECTIVE

Nitric oxide has been reported to reduce intimal hyperplasia as a response to arterial injury. This study was designed to assess the possible effect of perivascular application of a nitric oxide donor on neointimal proliferation occurring in veins exposed to the dynamics of the arterial circulation in a hypercholesterolemic rabbit model.

METHODS

Autologous jugular vein grafts were implanted in the carotid circulation of <em>20</em> hypercholesterolemic rabbits. A mixture of a biodegradable polymer and the nitric oxide donor, spermine/nitric oxide, which releases nitric oxide with a half-life of 39 minutes, was applied periadventitially at the time of implantation. Controls were veins bathed in saline solution, polymer alone, and polymer plus the carrier vehicle spermine without nitric oxide. Animals (n = 5 in each group) were put to death on day 28 for morphometric analysis, cell count, and immunohistochemical staining.

RESULTS

Treatment with perivascular nitric oxide donor significantly decreased wall thickness (126 +/- 24 microm vs <em>20</em>8 +/- 45 microm, p = 0.0017) and area (124 +/- 22 microm2/microm vs 211 +/- 37 microm2/microm, p = 0.005). With the carrier vehicle spermine alone, there was a trend toward reduced intimal thickness, but the change was not statistically significant. In the grafts treated with nitric oxide donor, expression of insulin-like growth factor, fibroblast growth factor, thrombospondins, fibronectin, and tenascin was reduced.

CONCLUSIONS

The periadventitial delivery of nitric oxide donor produces a reduction of neointimal hyperplasia in veins implanted in the arterial circulation. The mechanism of action is not entirely clear, but the reduction cannot be explained on the basis of decreased cell proliferation alone. Other possibilities are modulation of protein synthesis of vascular smooth muscle cells and production of extracellular matrix components.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) recently has been established as a survival- and transmitter-promoting neurotrophic agent for embryonic neurons in vitro. Its local application to lesioned adult optic and sciatic nerves has been shown to rescue axotomized retinal and sensory neurons that otherwise die. Following transection of the fimbria fornix pathway connecting the medial septum (MS) to the hippocampus, MS neurons undergo severe cell death, which can be prevented partially by infusion of nerve <em>growth</em> <em>factor</em> (NGF). In the same lesion paradigm, we find that 87% of these neurons visualized by cresyl-violet staining have disappeared by 4 weeks after unilateral fimbria fornix transection in adult rats. Implantation of gel foam soaked with 8 micrograms bFGF reduced neuron death to 68%. A similar rescue effect was seen with 0.3 microgram NGF. NGF administered at <em>20</em> micrograms reduced cell losses to 54%. Thus, bFGF rescued 22% and NGF at <em>20</em> micrograms 38% of the neurons that otherwise would have died. Choline acetyltransferase immunocytochemistry revealed dramatic losses of cholinergic neurons on the lesioned, compared with the unlesioned, side. Cholinergic neuron death was clearly reduced by the bFGF and NGF treatments. Basic FGF, in contrast to NGF, did not prevent a reduction in size of surviving neuronal cell bodies. Considered in the context of FGF being present in brain and hippocampal neurons, our results suggest a possible role for FGF as a neurotrophic <em>factor</em> for CNS neurons in vivo.

Clonally derived cultures of porcine skeletal muscle satellite cells were developed. The mitogenic effects of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), epidermal <em>growth</em> <em>factor</em> (EGF), insulin-like <em>growth</em> <em>factors</em> (IGF-I and -II), and platelet-derived <em>growth</em> <em>factors</em> (PDGF-AA and -BB) were examined. Individually, bFGF, IGFs, and PDGF-BB stimulated proliferation of porcine satellite cells grown in basal serum-free medium or Minimum Essential Medium containing 2% fetal bovine serum (MEM-2% FBS). EGF stimulated proliferation in MEM-2% FBS, but neither EGF nor PDGF-AA were mitogenic when added to serum-free medium. The interactions among bFGF, EGF, IGF-I, and PDGF-BB were examined in serum-free medium, using <em>growth</em> <em>factor</em> concentrations shown in dose-response experiments to induce maximal proliferative responses (10 ng/ml bFGF, EGF and PDGF-BB, and 50 ng/ml IGF-I). The combination of bFGF and IGF-I dramatically increased proliferation, and IGF-I also synergized with EGF to increase proliferation. EGF, IGF-I, and bFGF interacted with PDGF-BB to stimulate proliferation. With the exception of EGF and bFGF, combinations of two <em>growth</em> <em>factors</em> typically resulted in greater than additive responses. Simultaneous exposure of satellite cells to bFGF, PDGF-BB, EGF, and IGF-I produced a fivefold increase in DNA compared to cells grown in basal serum-free medium. Elimination of EGF did not reduce the mitogenic response, yet removal of IGF-I, bFGF, or PDGF-BB reduced proliferation by approximately 40, <em>20</em>, and 10%, respectively. These mitogens are likely physiological regulators of porcine satellite cell activity.

Epidermal <em>growth</em> <em>factor</em> receptor (EGFR) ligands are fundamental regulators of epithelial <em>growth</em>, differentiation, and neoplastic transformation. In addition to being potent mitogens for murine epidermal keratinocytes in vitro, transforming <em>growth</em> <em>factor</em> alpha (TGF alpha) and EGF elicit distinctive changes in keratin expression: Ca(2+)-mediated induction of the differentiation-specific keratins K1 and K10 is blocked, while simple epithelial keratins K8 and K18 are expressed aberrantly (C. Cheng et al., Cell <em>Growth</em>, & Differ., 4: 317-327, 1993). We have evaluated several additional <em>growth</em> <em>factors</em> to determine the specificity of this response for EGFR ligands. TGF alpha, keratinocyte <em>growth</em> <em>factor</em> (KGF), and acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF), but not basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) or insulin-like <em>growth</em> <em>factor</em> type I, block Ca(2+)-mediated expression of K1 while inducing K8. Since KGF and aFGF (but not bFGF) are ligands for the KGF receptor (KGFR), we explored the possibility that the TGF alpha/EGFR pathway is an intermediary in signaling through the KGFR. TGF alpha mRNA was increased in cells treated with KGF, aFGF, or TGF alpha but not bFGF or insulin-like <em>growth</em> <em>factor</em> type I. Similar changes were detected at the protein level; TGF alpha in conditioned medium (CM) from control, KGF-, TGF alpha-, and aFGF-treated cultures was 54 (+/- 8, SEM), 365 (+/- 50), 146 (+/- <em>20</em>), and 1<em>20</em> (+/- 50) pg/ml, respectively. KGF and TGF alpha also increased expression of cell-associated TGF alpha measured in keratinocyte lysates. KGF increased TGF alpha secretion and mRNA levels in human as well as mouse keratinocytes. CM from KGF-treated cultures stimulated cell <em>growth</em> when added to cultures of normal keratinocytes. Preincubation with neutralizing antibodies to both TGF alpha and KGF, but not KGF antibody alone, blocked cell <em>growth</em> in cultures treated with KGF CM, suggesting that the predominant keratinocyte mitogen in KGF CM is TGF alpha. In support of this hypothesis, treatment of keratinocytes for 5 min with either KGF CM or purified TGF alpha resulted in EGFR autophosphorylation. Furthermore, after approximately 24 h, KGF as well as TGF alpha induced EGFR down-regulation based on Western blot analysis and 125I-EGF binding. Induction of TGF alpha in KGF-treated keratinocytes, coupled to activation and down-modulation of the EGFR, suggests that TGF alpha may be a proximal effector of KGF action for at least certain aspects of epidermal <em>growth</em> and differentiation.

We have identified a class of transformed NIH3T3 mouse <em>fibroblasts</em> that arise at low frequencies in transfection experiments with DNA from both neoplastic and non-neoplastic cells and that may result from a low level of spontaneous transformation of NIH3T3 cells. DNA from the transformed cells was unable to transform NIH3T3 cells in a second cycle of transfection and, where examined, the cells showed no evidence for the uptake of the transfected DNA sequences. The results of Southern analyses demonstrate that a mouse homologue of the human met oncogene is amplified 4- to 8-fold in 7 of 10 lines of these transformed NIH3T3 mouse <em>fibroblasts</em>. The cells containing the amplified gene also exhibit at least a <em>20</em>-fold overexpression of an 8.5-kb mRNA that is homologous to met. To test the hypothesis that met encodes a <em>growth</em> <em>factor</em> receptor, we examined the binding of platelet-derived <em>growth</em> <em>factor</em>, epidermal <em>growth</em> <em>factor</em>, insulin-like <em>growth</em> <em>factor</em> I and gastrin-releasing peptide to transformed and non-transformed NIH3T3 cells. The results show that there is no significant elevation of the binding of these <em>growth</em> <em>factors</em> to cells containing amplification and overexpression of met.

BACKGROUND

Both benign and malignant growth of the prostate depend on the induction of a microvasculature. Basic fibroblast growth factor (bFGF), a potent angiogenic factor, is thought to play an important role in this process.

METHODS

bFGF expression in prostatic carcinoma was assessed by ELISA, reverse transcription polymerase chain reaction, and immunohistochemistry.

RESULTS

DU-145 and PC-3 tumor cells produced bFGF. Almost 80-90% of it was localized in the cytoplasm, and 10-20% was associated with extracellular matrix components. Immunohistochemical analysis of prostatic tissue sections showed that cancer cells stained more intensively as compared to putatively healthy epithelium. In prostate cancer patients, mean bFGF serum levels were significantly elevated when compared to a healthy control group (6.64 pg/ml vs. 1.28 pg/ml). Serum bFGF levels did not correlate with any other clinical marker such as PSA, tumor stage, or grade. Four out of five patients who progressed to a more advanced stage showed an increase in serum bFGF levels.

CONCLUSIONS

These results suggest that increased bFGF release may be associated with a more aggressive tumor phenotype.

Disruption of the regulatory communication from the stroma to the epithelium mediated by the FGF7/10-FGFR2 signaling axis in the prostate and expression of ectopic FGFR1 in prostatic epithelial cells often correlate with prostate cancer progression both in human and in experimental animals. Ectopic expression of constitutively active FGFR1 mutant (caFGFR1) at low levels in prostate epithelial cells induces low- to intermediate-grade prostatic intraepithelial neoplasia (PIN) within 6-8 months and high-grade PIN in <em>20</em>-25 months. Depression of the FGFR2 signaling in the prostate also disturbs homeostasis in the prostate and induces prostate hyperplasia. To study whether PIN lesions induced by the caFGFR1 were expression-level dependent, and whether expression of the caFGFR1 and depression of the FGFR2 signaling in the prostate synergistically disturbed prostate homeostasis, we generated two new strains of ARR2PBi-caFGFR1 transgenic mice, which highly expressed caFGFR1 in prostatic epithelial cells. The mice were crossed with KDNR mice to generate ARR2PBi-caFGFR1/KDNR bigenic mice. The ARR2PBi-caFGFR1 mice developed high-grade PIN within 8 months, which was significantly faster than the mice expressing caFGFR1 at low levels. In addition, depression of the FGFR2 signaling clearly promoted perturbation of cellular homeostasis induced by the caFGFR1. The results demonstrated that the PIN development in caFGFR1 transgenic mice was caFGFR1 dosage-dependent, and indicated that the ectopic FGFR1 and the resident FGFR2 in epithelial cells had opposite impacts on intercompartmental homeostasis in the prostate. The bigenic mice provide a model with cooperative aberrations in the <em>fibroblast</em> <em>growth</em> <em>factor</em> signaling axis for evaluation of tumor-initiating events in prostate tumorigenesis.

The effects of fetal bovine serum, insulin-like <em>growth</em> <em>factor</em>-I, and <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 on the regulation of the functional physical properties of adult bovine cartilage explants during an incubation period of 18-<em>20</em> days was determined, and the relationship between the measured functional properties of the cartilage and the tissue composition was assessed. Cartilage disks were tested in the uniaxial radially confined configuration by the application of low amplitude oscillatory displacement and measurement of the resultant load and streaming potential. For the control cartilage terminated just after explant, the modulus was 0.39 +/- 0.28 MPa, the open circuit hydraulic permeability was 2.0 +/- 1.0 x 10(-15) m2/(Pa.sec), and the electrokinetic (streaming potential) coefficient was -2.3 +/- 0.6 mV/MPa. Incubation of cartilage in medium supplemented with serum or insulin-like <em>growth</em> <em>factor</em>-I resulted in maintenance of the modulus and electrokinetic coefficient, whereas incubation in basal medium or medium supplemented with <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 led to a marked decrease from control values in the modulus and the amplitude of the electrokinetic coefficient. All of the culture conditions examined resulted in an increase in permeability that was not statistically significant. The variation in the electromechanical properties of all the cartilage samples tested was related to the density of tissue proteoglycan and collagen (hydroxyproline). The modulus was correlated with both the density of tissue proteoglycan (+0.014 MPa/[mg/ml]) and the density of tissue hydroxyproline (+0.008 MPa/[mg/ml]). The electrokinetic coefficient was also correlated with the density of proteoglycan (-0.080 [mV/MPa]/[mg/ml]) and the density of hydroxyproline (+0.064 [mV/MPa]/[mg/ml]). These data indicate that the regulation of chondrocyte matrix metabolism by <em>growth</em> <em>factors</em> can significantly affect the physical properties and function of cartilage.

OBJECTIVE

Cholesterol and lipoprotein metabolism display pronounced gender differences. Premenopausal women have lower LDL and higher HDL cholesterol, whereas men display higher synthetic rates of bile acids and cholesterol. The effects of the administration of exogenous hormones to humans and animals indicate that these gender differences can often be explained by estrogens. We evaluated how increased levels of endogenous estrogens modulate cholesterol and lipoprotein metabolism in women.

RESULTS

We studied healthy women during initiation of in vitro fertilization using blood samples obtained when endogenous estrogens were low and high. Cholesterol in VLDL and LDL, but not in HDL, was reduced <em>20</em>% when estrogens were high. Apolipoprotein B levels decreased 13%. Apolipoprotein A-I and triglyceride levels increased 8% and 37%, respectively, whereas lipoprotein(a) levels were unchanged. Circulating PCSK9, a suppressor of LDL receptors, was reduced 14% when estrogens were high. Serum markers of bile acid and cholesterol synthesis were unaltered. <em>Growth</em> hormone levels increased 3-fold when estrogens were high, whereas insulin-like <em>growth</em> <em>factor</em>-1 and <em>fibroblast</em> <em>growth</em> <em>factor</em>-21 concentrations were unaltered.

CONCLUSIONS

In women, Apolipoprotein B-containing particles and circulating PCSK9 are reduced when endogenous estrogens are high, indicating that endogenous estrogens induce hepatic LDL receptors partly through a posttranscriptional mechanism. However, estrogens do not stimulate bile acid or cholesterol synthesis.

Peroxisome proliferator activated receptors (PPARs) are a family of related receptors implicated in a diverse array of biological processes. There are 3 main isotypes of PPARs known as PPARalpha, PPARbeta and PPARgamma and each is organized into domains associated with a function such as ligand binding, activation and DNA binding. PPARs are activated by ligands, which can be both endogenous such as fatty acids or their derivatives, or synthetic, such as peroxisome proliferators, hypolipidaemic drugs, anti-inflammatory or insulin-sensitizing drugs. Once activated, PPARs bind to DNA and regulate gene transcription. The different isotypes differ in their expression patterns, lending clues on their function. PPARalpha is expressed mainly in liver whereas PPARgamma is expressed in fat and in some macrophages. Activation of PPARalpha in rodent liver is associated with peroxisome proliferation and with suppression of apoptosis and induction of cell proliferation. The mechanism by which activation of PPARalpha regulates apoptosis and proliferation is unclear but is likely to involve target gene transcription. Similarly, PPARgamma is involved in the induction of cell <em>growth</em> arrest occurring during the differentiation process of <em>fibroblasts</em> to adipocytes. However, it has been implicated in the regulation of cell cycle and cell proliferation in colon cancer models. Less in known concerning PPARbeta but it was identified as a downstream target gene for APC/beta-catenin/T cell <em>factor</em>-4 tumor suppressor pathway, which is involved in the regulation of <em>growth</em> promoting genes such as c-myc and cyclin D1. Marked species and tissue differences in the expression of PPARs complicate the extrapolation of pre-clinical data to humans. For example, PPARalpha ligands such as the hypolipidaemic fibrates have been used extensively in the clinic over the past <em>20</em> years to treat cardiovascular disease and side effects of clinical fibrate use are rare, despite the observation that these compounds are rodent carcinogens. Similarly, adverse clinical responses have been seen with PPARgamma ligands that were not predicted by pre-clinical models. Here, we consider the response to PPAR ligands seen in pre-clinical models of efficacy and safety in the context of human health and disease.

The method of affinity coelectrophoresis was used to study the binding of nine representative glycosaminoglycan (GAG)-binding proteins, all thought to play roles in nervous system development, to GAGs and proteoglycans isolated from developing rat brain. Binding to heparin and non-neural heparan and chondroitin sulfates was also measured. All nine proteins-laminin-1, fibronectin, thrombospondin-1, NCAM, L1, protease nexin-1, urokinase plasminogen activator, thrombin, and <em>fibroblast</em> <em>growth</em> <em>factor</em>-2-bound brain heparan sulfate less strongly than heparin, but the degree of difference in affinity varied considerably. Protease nexin-1 bound brain heparan sulfate only 1.8-fold less tightly than heparin (Kdvalues of 35 vs. <em>20</em> nM, respectively), whereas NCAM and L1 bound heparin well (Kd approximately 140 nM) but failed to bind detectably to brain heparan sulfate (Kd>3 microM). Four proteins bound brain chondroitin sulfate, with affinities equal to or a few fold stronger than the same proteins displayed toward cartilage chondroitin sulfate. Overall, the highest affinities were observed with intact heparan sulfate proteoglycans: laminin-1's affinities for the proteoglycans cerebroglycan (glypican-2), glypican-1 and syndecan-3 were 300- to 1800-fold stronger than its affinity for brain heparan sulfate. In contrast, the affinities of <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 for cerebroglycan and for brain heparan sulfate were similar. Interestingly, partial proteolysis of cerebroglycan resulted in a >400-fold loss of laminin affinity. These data support the views that (1) GAG-binding proteins can be differentially sensitive to variations in GAG structure, and (2) core proteins can have dramatic, ligand-specific influences on protein-proteoglycan interactions.

Synovial <em>fibroblasts</em> are likely to be a significant source of granulocyte-macrophage colony-stimulating <em>factor</em> (GM-CSF) and granulocyte-CSF (G-CSF), which could be crucial to the pathogenesis of rheumatoid arthritis. Using specific enzyme-linked immunosorbent assays (ELISAs) and Northern analysis, GM-CSF and G-CSF expression were followed in human synovial <em>fibroblast</em>-like cells in response to a number of agents, either alone or in the presence of an optimal stimulatory concentration of interleukin-1 (IL-1). For both CSFs, interferon-gamma (100 U/mL) did not increase their levels but dramatically suppressed the stimulatory action of IL-1, while basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (10(-8) mol/L), although nonstimulatory by itself, potentiated IL-1 action. The glucocorticoid, dexamethasone (10(-7) mol/L), inhibited IL-1-stimulated CSF production. However, evidence was obtained for noncoordinated CSF regulation. Cyclooxygenase inhibitors potentiated the action of IL-1 on GM-CSF synthesis but suppressed G-CSF synthesis, suggesting that endogenous cyclooxygenase products can have opposite effects in modulating the levels of each CSF. Also, the lymphokine, IL-4 (250 pmol/L), slightly inhibited GM-CSF formation in the presence of IL-1 but elevated the G-CSF levels in these cultures without having an effect by itself. Transforming <em>growth</em> <em>factor</em> beta (less than or equal to <em>20</em> ng/mL) did not modulate levels of either CSF. Mesenchymal cell production of both GM-CSF and G-CSF is generally viewed as being under coordinate control; our findings suggest that their synthesis in IL-1-stimulated human synoviocytes can be modulated by a number of agents, in some cases with divergent actions depending on which CSF is examined.

Compared to basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), a widely distributed, broad spectrum mitogen and mesoderm inducer, acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) is reported to have an essentially neural distribution and to be undetectable in the early embryo. In the present investigation, we used immunoblotting and immunochemistry to assess the cellular and tissue distributions of aFGF and bFGF in 11-<em>20</em>-d rat embryos. Immunoblotting of crude and heparin-bound embryo extracts revealed faint bands at the expected 17-18-kD and predominant bands at an apparent molecular mass of 26 to 28-kD (despite reducing conditions) using multiple specific antibodies for aFGF and bFGF. Pretreatment with 8 M urea yielded 18-<em>20</em>-kD aFGF and bFGF and some 24-26-kD bFGF. Immunoreactivity for both aFGF and bFGF was positive and similar in the cytoplasm, nuclei, and extracellular matrix of cells of neuroectodermal and mesodermal origin, while it was negative in endoderm-derived cells. The distribution of immunoreactive aFGF and bFGF also showed changes during development that were associated with the process of cellular and tissue differentiation. For example, intensity and extent of immunoreactivity for both peptides progressively increased in the middle layer of the spinal cord with increasing differentiation of the neural cells. The immunostaining patterns were very similar for aFGF and bFGF for each organ and at each stage. In conclusion, high molecular mass forms of immunoreactive aFGF and bFGF are present in the rat embryo. Acidic FGF and bFGF are both widely distributed in tissues of neuroectodermal and mesodermal origin, and their distribution was very similar.

OBJECTIVE

Maternal food restriction during pregnancy results in growth-restricted newborns and reduced glomerular number, contributing to programmed offspring hypertension. We investigated whether reduced nephrogenesis may be programmed by dysregulation of factors controlling ureteric bud branching and mesenchyme to epithelial transformation.

METHODS

At 10 to 20 days' gestation, Sprague Dawley pregnant rats (n = 6/group) received ad libitum food; food-restricted rats were 50% food restricted. At embryonic day 20, messenger ribonucleic acid (mRNA) and protein expression of Wilms' tumor 1 gene product (WT1), paired box transcription factor (Pax)-2, fibroblast growth factor (FGF)-2, glial cell line-derived neurotrophic factor (GDNF), cRET, wingless-type mouse mammary tumor virus integration site (WNT)4, WNT11, bone morphogenetic protein (BMP)-4, BMP7, and FGF7 were determined by real-time polymerase chain reaction and Western blotting.

RESULTS

Maternal food restriction resulted in up-regulated mRNA expression for WT1, FGF2, and BMP7, whereas Pax2, GDNF, FGF7, BMP4, WNT4, and WNT11 mRNAs were down-regulated. Protein expression was concordant for WT1, GDNF, Pax2, FGF7, BMP4, and WNT4.

CONCLUSIONS

Maternal food restriction altered gene expression of fetal renal transcription and growth factors and likely contributes to development of offspring hypertension.

Human embryonic stem (hES) cells have traditionally been cultured in medium containing fetal calf serum (FCS) and mouse <em>fibroblasts</em> as feeder cells. The use of animal derived materials carries a risk of transmitting animal pathogens, and they are not optimal in cultures aimed at cell transplantation in humans. This technical study aiming at facilitating IVF units to establish new hES cell lines, has systematically compared the non-differentiated <em>growth</em> of the hES cell line HS237, originally derived and thereafter cultured using human foreskin <em>fibroblasts</em> as feeder cells, by culturing it in media containing serum replacement (SR; 10, 15, <em>20</em>%), FCS, and human serum. In addition, optimal concentrations of insulin-transferrin-selenium (ITS) mixture and the effect of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) have also been studied. Cellular <em>growth</em> was monitored daily and maintenance of their non-differentiated character was studied using antibodies against TRA-1-60, TRA-1-81 and SSEA-4 and expression of Oct-4. The hES cells proliferated fastest when <em>20</em>% of SR was used. In human serum-containing medium, the cells underwent extensive spontaneous differentiation within a few passages. The FCS supported the non-differentiated <em>growth</em> poorly. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> supported non-differentiated <em>growth</em>, the highest concentration (8 ng/ml) giving the best result, while ITS was not beneficial.

Uncovering the origin and nature of phenotypic variation within species is the first step in understanding variation between species. Mouse models with altered activities of crucial signal pathways have highlighted many important genes and signal networks regulating the morphogenesis of complex structures, such as teeth. The detailed analyses of these models have indicated that the balanced actions of a few pathways regulating cell behavior modulate the shape and number of teeth. Currently, however, most mouse models studied have had gross alteration of morphology, whereas analyses of more subtle modification of morphology are required to link developmental studies to evolutionary change. Here, we have analyzed a signaling network involving ectodysplasin (Eda) and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>20</em> (Fgf<em>20</em>) that subtly affects tooth morphogenesis. We found that Fgf<em>20</em> is a major downstream effector of Eda and affects Eda-regulated characteristics of tooth morphogenesis, including the number, size and shape of teeth. Fgf<em>20</em> function is compensated for by other Fgfs, in particular Fgf9 and Fgf4, and is part of an Fgf signaling loop between epithelium and mesenchyme. We showed that removal of Fgf<em>20</em> in an Eda gain-of-function mouse model results in an Eda loss-of-function phenotype in terms of reduced tooth complexity and third molar appearance. However, the extra anterior molar, a structure lost during rodent evolution 50 million years ago, was stabilized in these mice.

Repair of the vascular lumenal surface after injury requires a controlled endothelial cell response that includes cell migration, proliferation, and remodeling of the extracellular matrix. These cellular processes are modulated by <em>growth</em> <em>factors</em> that are released or activated following cell injury. When endothelial cell migration is stimulated in response to monolayer wounding in vitro, cells increase synthesis of small leucine-rich dermatan sulfate proteoglycans (PGs) (Kinsella, M. G., and Wight, T. N. (1986) J. Cell Biol. 102, 679-687). However, the identity of the PGs that are increased during cell migration and the <em>factors</em> that affect this modulation have not been identified. We now report that basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is responsible for the transient increase of [35S]sulfate incorporation into PGs following monolayer wounding. SDS-polyacrylamide gel electrophoresis analysis revealed that bFGF-treated and wounded cultures increase both biglycan core protein synthesis and biglycan proteolytic processing, which results in the accumulation of a approximately <em>20</em>-kDa N-terminal biglycan fragment in the culture media. Biglycan RNA steady-state levels also selectively increase 2- to 3-fold after wounding or bFGF treatment. Finally, immunocytochemical staining localizes biglycan to the tips and edges of lamellopodia on migrating cells, indicating that biglycan is found at loci at which the formation and dissolution of adhesion plaques occurs, consistent with hypotheses that predict involvement of biglycan in the control of cell migration. Taken together, these results suggest that release of endogenous bFGF is primarily responsible for altered biglycan expression, synthesis, and proteolytic processing as endothelial cells migrate after wounding.