Large segmental bone defect repair remains a clinical and scientific challenge with increasing interest focused on combining gene transfer with tissue engineering techniques. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is one of the most prominent osteogenic <em>growth</em> <em>factors</em> that has the potential to accelerate bone healing by promoting the proliferation and differentiation of mesenchymal stem cells (MSCs) and the regeneration of capillary vasculature. However, the short biological half-lives of <em>growth</em> <em>factors</em> may impose severe restraints on their clinical usefulness. Gene-based delivery systems provide a better way of achieving a sustained high concentration of <em>growth</em> <em>factors</em> locally in the defect and delivering a more biologically active product than that achieved by exogenous application of recombinant proteins. The objective of this experimental study was to investigate whether the bFGF gene modified MSCs could enhance the repair of large segmental bone defects. The pcDNA3-bFGF gene transfected MSCs were seeded on biodegradable porous beta tricalcium phosphate (beta-TCP) ceramics and allografted into the <em>15</em> mm critical-sized segmental bone defects in the radius of 18 New Zealand White rabbits. The pcDNA3 vector gene transfected MSCs were taken as the control. The follow-up times were 2, 4, 6, 8, 10 and 12 weeks. Scanning electron microscopic, roentgenographic, histologic and immunohistological studies were used to assess angiogenesis and bone regeneration. In vitro, the proliferation and differentiation of bFGF gene transfected MSCs were more active than that of the control groups. In vivo, significantly more new bone formation accompanied by abundant active capillary regeneration was observed in pores of the ceramics loaded with bFGF gene transfected MSCs, compared with control groups. Transfer of gene encoding bFGF to MSCs increases their osteogenic properties by enhancing capillary regeneration, thus providing a rich blood supply for new bone formation. This new bFGF gene enhanced tissue engineering strategy could be of potential benefit to accelerate bone healing, especially in defects caused by atrophic nonunion and avascular necrosis of the femoral head.

In Alzheimer's disease, abnormal extracellular accumulations of beta-amyloid (a major component of the senile plaques) and of the excitatory amino acid glutamate are both thought to be associated with degeneration of nerve cells. In the present study, using cultured cortical or hippocampal neurons as an in vitro model, we compared the effects of various <em>factors</em> influencing neurodegeneration mediated by glutamate or by beta-amyloid peptide (A beta). We also asked the question: does long-term treatment with sublethal doses of A beta-(25-35) potentiate glutamate-mediated excitotoxicity? Neuronal cell death was quantified using the lactate dehydrogenase (LDH) method. Since extracellular LDH remains stable for days, the magnitude of relative afflux of LDH correlates in a linear fashion with the number of damaged neurons in cultures. When applied singly, both glutamate (for <em>15</em> min) and A beta-(25-35) or its parent peptide A beta-(1-40) (continuously) produced a dose-dependent neuronal degeneration. In the case of glutamate, the half-maximal effects were observed at about 0.08 mM glutamate for both cerebral cortical and hippocampal neurons (cultured for 13 days in vitro, DIV). The effect of A beta-(25-35) was also time-dependent, while neurons grown in a chemically defined medium showed relatively greater susceptibility to A beta-(25-35) than those cultured in a serum-containing medium. These differential effects were not related to the presence of different numbers of glial cells in the cultures. Treatment with different doses of the antimitotic inhibitor, cytosine arabinoside, for 24 h (6-7 DIV) produced at 13 DIV cortical neuronal cultures with varying numbers of astrocytes, as determined by the astrocyte-specific enzyme glutamine synthetase. The presence of astrocytes decreased the toxicity of glutamate for neurons. The modulation was due to uptake of glutamate by astrocytes, thereby reducing its effective concentration, as the effect was seen at 0.1 mM and not at 10 mM glutamate. Incorporation of an NMDA receptor mediated Ca2+ ion channel blocker, MK-801, together with glutamate completely inhibited degeneration of cortical neurons, and pretreatment of cultures with basic <em>fibroblast</em> <em>growth</em> <em>factor</em> for 2 days did so partially. However, these compounds had no effect on neurotoxicity mediated by A beta-(25-35). Lastly, the effect of glutamate interacted with that of A beta-(25-35). Pretreatment of cortical neurons for 2 days with 10 microM A beta-(25-35) by itself had no appreciable effect, but it potentiated significantly the degeneration of these neurons mediated by glutamate.(ABSTRACT TRUNCATED AT 400 WORDS)

The protein tyrosine phosphatase 1B (PTP1B) modulates tyrosine kinase receptors, among which is the vascular endothelial <em>growth</em> <em>factor</em> receptor type 2 (VEGFR2), a key component of angiogenesis. Because PTP1B deficiency in mice improves left ventricular (LV) function 2 mo after myocardial infarction (MI), we hypothesized that enhanced angiogenesis early after MI via activated VEGFR2 contributes to this improvement. At 3 d after MI, capillary density was increased at the infarct border of PTP1B(-/-) mice [+7±2% vs. wild-type (WT), P = 0.05]. This was associated with increased extracellular signal-regulated kinase 2 phosphorylation and VEGFR2 activation (i.e., phosphorylated-Src/Src/VEGFR2 and dissociation of endothelial VEGFR2/VE-cadherin), together with higher infiltration of proangiogenic M2 macrophages within unchanged overall infiltration. In vitro, we showed that PTP1B inhibition or silencing using RNA interference increased VEGF-induced migration and proliferation of mouse heart microvascular endothelial cells as well as <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-induced proliferation of rat aortic smooth muscle cells. At 8 d after MI in PTP1B(-/-) mice, increased LV capillary density (+21±3% vs. WT; P<0.05) and an increased number of small diameter arteries (<em>15</em>-50 μm) were likely to participate in increased LV perfusion assessed by magnetic resonance imaging and improved LV compliance, indicating reduced diastolic dysfunction. In conclusion, PTP1B deficiency reduces MI-induced heart failure promptly after ischemia by enhancing angiogenesis, myocardial perfusion, and diastolic function.

Achondroplasia (ACH) is the most frequent form of dwarfism and is caused by gain-of-function mutations in the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3-encoding (FGFR3-encoding) gene. Although potential therapeutic strategies for ACH, which aim to reduce excessive FGFR3 activation, have emerged over many years, the use of tyrosine kinase inhibitor (TKI) to counteract FGFR3 hyperactivity has yet to be evaluated. Here, we have reported that the pan-FGFR TKI, NVP-BGJ398, reduces FGFR3 phosphorylation and corrects the abnormal femoral <em>growth</em> plate and calvaria in organ cultures from embryos of the Fgfr3Y367C/+ mouse model of ACH. Moreover, we demonstrated that a low dose of NVP-BGJ398, injected subcutaneously, was able to penetrate into the <em>growth</em> plate of Fgfr3Y367C/+ mice and modify its organization. Improvements to the axial and appendicular skeletons were noticeable after 10 days of treatment and were more extensive after <em>15</em> days of treatment that started from postnatal day 1. Low-dose NVP-BGJ398 treatment reduced intervertebral disc defects of lumbar vertebrae, loss of synchondroses, and foramen-magnum shape anomalies. NVP-BGJ398 inhibited FGFR3 downstream signaling pathways, including MAPK, SOX9, STAT1, and PLCγ, in the <em>growth</em> plates of Fgfr3Y367C/+ mice and in cultured chondrocyte models of ACH. Together, our data demonstrate that NVP-BGJ398 corrects pathological hallmarks of ACH and support TKIs as a potential therapeutic approach for ACH.

Cumulative data suggest that neuroinflammation plays a prominent role in Alzheimer's disease (AD) pathogenesis. The purpose of this work was to assess if patients with AD present a specific cerebrospinal fluid (CSF) cytokine profile and if it correlates to disease progression. We determined the levels of 27 cytokines in CSF of patients with AD and compared them with patients with frontotemporal dementia and nondemented controls. In addition, we correlated the cytokine levels with cognitive status and disease progression after 12 months. Patients with AD had higher levels of proinflammatory and anti-inflammatory cytokines (eotaxin, interleukin [IL]-1ra, IL-4, IL-7, IL-8, IL-9, IL-10, IL-<em>15</em>, granulocyte colony-stimulating <em>factor</em>, monocyte chemotactic protein 1, platelet-derived <em>growth</em> <em>factor</em>, tumor necrosis <em>factor</em> alfa) compared to nondemented controls. There was a negative correlation between the disease progression and the levels of several cytokines (IL-1β, IL-4, IL-6, IL-9, IL-17A, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, granulocyte colony-stimulating <em>factor</em>, granulocyte-macrophage colony-stimulating <em>factor</em>, interferon gamma, macrophage inflammatory proteins-1β). To the best of our knowledge, this is the first study reporting a "protective" role of the upregulation of specific intrathecal cytokine levels in AD. This finding supports that a fine "rebalancing" of the immune system represents a new target in AD therapeutic approach.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the Western world, and safe and effective therapies are needed. Bile acids (BAs) and their receptors [including the nuclear receptor for BAs, farnesoid X receptor (FXR)] play integral roles in regulating whole-body metabolism and hepatic lipid homeostasis. We hypothesized that interruption of the enterohepatic BA circulation using a luminally restricted apical sodium-dependent BA transporter (ASBT) inhibitor (ASBTi; SC-435) would modify signaling in the gut-liver axis and reduce steatohepatitis in high-fat diet (HFD)-fed mice. Administration of this ASBTi increased fecal BA excretion and messenger RNA (mRNA) expression of BA synthesis genes in liver and reduced mRNA expression of ileal BA-responsive genes, including the negative feedback regulator of BA synthesis, <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em>. ASBT inhibition resulted in a marked shift in hepatic BA composition, with a reduction in hydrophilic, FXR antagonistic species and an increase in FXR agonistic BAs. ASBT inhibition restored glucose tolerance, reduced hepatic triglyceride and total cholesterol concentrations, and improved NAFLD activity score in HFD-fed mice. These changes were associated with reduced hepatic expression of lipid synthesis genes (including liver X receptor target genes) and normalized expression of the central lipogenic transcription <em>factor</em>, Srebp1c Accumulation of hepatic lipids and SREBP1 protein were markedly reduced in HFD-fed Asbt(-/-) mice, providing genetic evidence for a protective role mediated by interruption of the enterohepatic BA circulation. Together, these studies suggest that blocking ASBT function with a luminally restricted inhibitor can improve both hepatic and whole body aspects of NAFLD.

Fibrotic disorders of the renal, pulmonary, cardiac, and hepatic systems are associated with significant morbidity and mortality. Effective therapies to prevent or curtail the advancement to organ failure, however, remain a major clinical challenge. Chronic kidney disease, in particular, constitutes an increasing medical burden affecting>><em>15</em>% of the US population. Regardless of etiology (diabetes, hypertension, ischemia, acute injury, urologic obstruction), persistently elevated TGF-β1 levels are causatively linked to the activation of profibrotic signaling networks and disease progression. TGF-β1 is the principal driver of renal fibrogenesis, a dynamic pathophysiologic process that involves tubular cell injury/apoptosis, infiltration of inflammatory cells, interstitial <em>fibroblast</em> activation and excess extracellular matrix synthesis/deposition leading to impaired kidney function and, eventually, to chronic and end-stage disease. TGF-β1 activates the ALK5 type I receptor (which phosphorylates SMAD2/3) as well as non-canonical (e.g., src kinase, EGFR, JAK/STAT, p53) pathways that collectively drive the fibrotic genomic program. Such multiplexed signal integration has pathophysiological consequences. Indeed, TGF-β1 stimulates the activation and assembly of p53-SMAD3 complexes required for transcription of the renal fibrotic genes plasminogen activator inhibitor-1, connective tissue <em>growth</em> <em>factor</em> and TGF-β1. Tubular-specific ablation of p53 in mice or pifithrin-α-mediated inactivation of p53 prevents epithelial G2/M arrest, reduces the secretion of fibrotic effectors and attenuates the transition from acute to chronic renal injury, further supporting the involvement of p53 in disease progression. This review focuses on the pathophysiology of TGF-β1-initiated renal fibrogenesis and the role of p53 as a regulator of profibrotic gene expression.

BACKGROUND

Aqueous flare as determined by laser flare photometry in the anterior chamber is a strong preoperative predictor for proliferative vitreoretinopathy (PVR) in patients with primary retinal detachment (RD). We analysed various cytokines in aqueous humour samples in relation to aqueous flare and postoperative PVR incidence in patients with RD.

METHODS

Preoperatively, the aqueous flare of patients with RD was measured quantitatively with a laser flare metre and aqueous humour samples were collected and analysed for interferon γ, tumour necrosis factor α, monocyte chemoattractant protein (MCP)-1, interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, vascular endothelial growth factor (VEGF)-A, platelet derived growth factor (PDGF)-aa, transforming growth factor (TGF)-β1, TGF-β2, TGF-β3, fibroblast growth factor (FGF)-aa and FGF-bb by multiplex fluorescent bead-based immunoassays. Three months after RD surgery patients were examined for PVR development.

RESULTS

Of 67 consecutive patients, 10 developed at least PVR grade C. Patients with flare values >15 pc/ms (n=20) and the 10 patients with postoperative PVR all had significantly elevated levels of IL-6, IL-8, MCP-1 and TGF-β1 in aqueous humour (p≤0.05). Levels of VEGF-A, PDGF-aa and TGF-β2 were not significantly changed. Other cytokines were below the detection threshold. Eight of the 10 patients (80%) with PVR had elevated flare values of >15 pc/ms and 8 of the 20 patients (40%) with flare >15 pc/ms developed PVR. The OR for PVR with flare values >15 pc/ms was 30.7 (p=0.0001).

CONCLUSIONS

Laser flare photometry allows simple risk estimation for later PVR development. Elevated laser flare values correspond to an altered profibrotic intraocular cytokine milieu. These factors therefore constitute promising targets for a prophylactic intervention.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) regulate many important developmental and homeostatic physiological events. The FGF superfamily contains several families. In this review, we present recent findings on the two FGFs of the nematode Caenorhabditis elegans from both functional and phylogenic points of view. C. elegans has a single FGFR (EGL-<em>15</em>) with two functionally exclusive isoforms, and two FGFs (LET-756 and EGL-17), which play distinct roles: an essential function for the former, and guidance of the migrating sex myoblasts for the latter. Regulation of homeostasis by control of the fluid balance could be the basis for the essential function of LET-756. Phylogenetic and functional studies suggest that LET-756, like vertebrate FGF9, -16, and -20, belongs to the FGF9 family, whereas EGL-17, like vertebrate FGF8, -17, and -18, could be included in the FGF8 family.

MicroRNA-126 (miR-126), an endothelial-specific miRNA located within intron 7 of epidermal <em>growth</em> <em>factor</em>‑like domain 7 (EGFL7), has been demonstrated to act as a tumor suppressor in various types of human cancer. However, its role in oral squamous cell carcinoma (OSCC) remains unclear. In the present study, we revealed that the expression of miR-126 was significantly decreased in OSCC tissues, when compared with that in their matched adjacent tissues, and its expression level was also reduced in Tca8113, OSCC-<em>15</em> and CAL27 cell lines compared with normal tissues. The protein expression of EGFL7 was upregulated in OSCC tissues compared with their matched adjacent tissues as well as normal tissues, and Tca8113, OSCC-<em>15</em> and CAL27 cells additionally demonstrated a positive expression of EGFL7. The overexpression of miR-126 significantly reduced the protein expression of EGFL7 in OSCC-<em>15</em> cells, while transfection with the miR-126 inhibitor upregulated the EGFL7 protein level in OSCC-<em>15</em> cells. Furthermore, transfection with an miR-126 mimic into OSCC-<em>15</em> cells markedly suppressed cell proliferation, cell cycle progression, cell invasion and colony formation, while inducing cell apoptosis, which contrasted with the effects of transfection with an miR-126 inhibitor. The overexpression of miR-126 suppressed the secretion of two key regulators of angiogenesis, vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), which was also reversed by miR-126 inhibitor transfection. In conclusion, the present study demonstrated that miR-126 acts as a tumor suppressor in OSCC cells, partially at least via the downregulation of EGFL7. Thus, miR-126 may serve as a promising candidate for the treatment of OSCC.

In pulmonary hypertension, induced in rats breathing high oxygen at normobaric pressure, vascular cell hypertrophy and hyperplasia thicken the walls of lung microvessels (<em>15</em>-100 microns in diameter). Over a 28-day time course, new contractile cells develop from intimal precursor smooth muscle cells, which include intermediate cells and interstitial <em>fibroblasts</em>. Cell labeling studies in vivo have shown that these cells proliferate more than other vascular cells and that most of this activity occurs between 4 and 7 days of hyperoxia. The <em>growth</em> <em>factors</em> responsible for this proliferation are unknown. In the present study, we investigate the expression of mRNA for the epidermal <em>growth</em> <em>factor</em> (EGF)-related protein, heparin-binding EGF-like <em>growth</em> <em>factor</em> (HB-EGF), a newly discovered mitogen for <em>fibroblasts</em> and smooth muscle cells. Northern analysis shows HB-EGF mRNA levels to be low in normal lung but increased 100-fold by day 7 of hyperoxia. In situ hybridization identifies a select group of cells expressing HB-EGF mRNA. In normal lung, hybridizing cells are randomly distributed in the alveolar wall and space. By day 7, they increase in number and cluster around the microvessels. Histochemical techniques identify cells expressing HB-EGF mRNA as eosinophils.

Plasma membrane protrusion is fundamental to cell motility, but its regulation by the extracellular environment is not well elucidated. We have quantified lamellipodial protrusion dynamics in human vascular smooth muscle cells exposed to <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF-2) and type I collagen, two distinct ligands presented to vascular cells during arterial remodeling. Video microscopy revealed that FGF-2 stimulated a modest increase in lamellipodial protrusion rate that peaked within <em>15</em> min. This response was associated with immediate but transient activation of Rac1 and was inhibited in cells infected with retrovirus containing cDNA encoding dominant-negative Rac1. A 1-h exposure to FGF-2 also set up a second phase of more striking lamellipodial protrusion evident at 24-36 h. This delayed response was most pronounced when cells were on type 1 collagen and was associated with FGF-2-induced expression of collagenase-1 that localized to the edge of protruding lamellipodia. Moreover, late membrane protrusion was inhibited when cells were on collagenase-resistant type I collagen, implicating degraded collagen as a mediator. For cells on collagen, the immediate activation of Rac1 by FGF-2 was followed by a sustained wave of Rac1 activation that was inhibited when cleavage of the collagen triple helix was prevented and also by blockade of alpha(v)beta(3) integrin. We conclude that lamellipodial protrusion in smooth muscle cells can be regulated by waves of Rac1 activation, corresponding to the sequential presentation of FGF-2 and remodeled collagen. The findings thus reveal a previously unrecognized level of coordination among extracellular input that enables cells to maintain protrusive activity over prolonged periods.

PC cell derived <em>growth</em> <em>factor</em> (PCDGF) is an 88-kDa glycoprotein purified from the culture medium of the highly tumorigenic mouse teratoma-derived cell line PC. PCDGF was shown to stimulate the proliferation of 3T3 <em>fibroblasts</em> and PC cells. Amino acid sequencing of PCDGF indicated its identity to the precursor for the 6-kDa polypeptides epithelins and granulins. In this paper, we investigated the binding of PCDGF to the mink lung epithelial cell line CCL64. Scatchard analysis indicates that 125I-PCDGF binding to CCL64 cells is curvilinear, corresponding to the existence of two classes of binding sites: high affinity binding sites (560 +/- 170 sites/cell) with a Kd1 of 43 +/- <em>15</em> pM and low affinity binding sites (16,350 +/- 5900 sites/cell) with a Kd2 of 3.9 +/- 1.9 nM. 125I-PCDGF was chemically crosslinked to cell surface receptors on CCL64 cells with disuccinimidyl suberate. A major crosslinked band of about 190 kDa with radiolabeled PCDGF was detected after SDS-PAGE, suggesting the presence of PCDGF binding sites with molecular weight of about 120 kDa. 125I. PCDGF crosslinking studies indicate the presence of PCDGF binding sites with a molecular weight similar to those of binding sites on CCL64 cells on the surface of two other PCDGF-responsive cell lines, 3T3 <em>fibroblasts</em> and PC cells. These data suggest that the receptors for PCDGF are widely distributed on cells of distinct embryonic origin.

After the debridement of the entire corneal epithelium of the rabbit eye, epithelial cells of conjunctival origin cover the denuded corneal surface. Under such experimental conditions, the rate of wound healing is considerably delayed and total regeneration is completed within <em>15</em> to 20 days, allowing evaluation of various drugs, such as the <em>Fibroblast</em> <em>Growth</em> <em>Factor</em>. Both acidic and basic FGF were administered topically on totally de-epithelialized rabbit eye, at three different concentrations of 1.5 and 10 Stimulation Units/50 microliters, 3 times per day. A dose-response effect was observed and in each case, acidic FGF was found to be much more potent than bFGF in increasing the rate of wound healing of the cornea. These results are correlated with a new purification procedure, avoiding acid treatment of the tissue extract. The systemic diffusion of FGF allows the contralateral eye cells to be also stimulated for mitosis and migration and to heal faster than the control eyes.

We have previously reported that basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) can induce retinal regeneration in the stage 22-24 chicken embryo. The present study was undertaken to identify the cellular source of the regenerate and to determine whether other <em>growth</em> <em>factors</em> also elicit regeneration in this animal model. Polymer implants containing bFGF were inserted into eyes of chicken embryos immediately after extirpation of the neural retina. The retinal pigment epithelium (RPE) was left intact. Evaluation by light microscopy revealed that in bFGF-treated eyes the new neural retina arose by transdifferentiation of the entire RPE layer. Differentiation of the new neural retina occurred in a sequence similar to that of normal development but proceeded in a reverse (vitread) direction. All retinal laminae had differentiated by Day <em>15</em>. However, the regenerate displayed reversed polarity, with photoreceptors closest to the lens. The RPE, pecten, and optic nerve were absent. Focal areas of degeneration in the retinal regenerate became evident for the first time on Day 10. Retinal regeneration was also observed after treatment with higher doses of acidic <em>fibroblast</em> <em>growth</em> <em>factor</em>, but not with nerve <em>growth</em> <em>factor</em>-beta, transforming <em>growth</em> <em>factor</em>-beta 1, insulin, or insulin-like <em>growth</em> <em>factors</em> I or II. These results raise the possibility that FGFs may play a role in retinal differentiation during development.

Angiogenesis is controlled by a balance between stimulatory <em>growth</em> <em>factors</em> and endogenous inhibitors. We propose that the balance of stimulators and inhibitors, as well as the general sensitivity of the endothelium to these <em>factors</em>, varies from individual to individual. Indeed, we have found that individual mouse strains have dramatically different responses to <em>growth</em> <em>factor</em>-induced neovascularization. Quantitative trait loci (QTLs), which influence the extent of angiogenesis induced by vascular endothelial <em>growth</em> <em>factor</em> (VEGF), were previously identified by our laboratory. Since genetic susceptibility may vary according to the angiogenic stimulator, we have undertaken a similar mapping approach to identify QTLs that influence basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF2) induced neovascularization in the BXD series of recombinant inbred mouse strains. Composite and multiple interval mapping identified areas of chromosomes 4, 13, <em>15</em>, and 18. These new angiogenesis QTLs, named AngFq1 through AngFq4 (for angiogenesis due to FGF2), are different from previously identified VEGF QTLs. The mapped regions contain several genes involved in the angiogenic process including matrix metalloproteinase 16, eph receptor A7, angiopoetin 1, endothelial lipase, and autotaxin. Differences in these regions may influence individual susceptibility to angiogenesis related diseases such as cancer, macular degeneration, atherosclerosis, and arthritis.

OBJECTIVE

Interleukin-6 is a major trigger for the synthesis of acute phase proteins by liver parenchymal cells. Acute phase proteins may contribute to the regulation of liver fibrosis by inhibition of proteases (e.g. collagenase) and by binding of cytokines. Since liver (myo)fibroblasts play an important role in the production of extracellular matrix in fibrotic livers, a study was undertaken into whether these cells are able to synthesize interleukin-6, which would give them the opportunity to contribute to regulation of synthesis of acute phase proteins by neighbouring parenchymal cells.

METHODS

In the present study we investigated interleukin-6 production by two cell types obtained from human liver tissue: human fat-storing cells obtained from 5-15% Percoll fractions, which transformed in culture into myofibroblasts co-expressing alpha-smooth muscle actin and vimentin (VA cells) and fibroblasts obtained from 30-40% Percoll fractions which express vimentin only (V vells). Interleukin-6 production was measured in culture media of these cells using an enzyme-linked immunosorbent assay after incubation with lipopolysaccharide, and mediators like interleukin-1 beta, tumor necrosis factor-alpha, transforming growth factor-beta and interferon-gamma, known to be present in elevated concentrations in fibrotic livers.

RESULTS

Unstimulated human liver (myo)fibroblasts produced considerable amounts of interleukin-6 (287 ng/mg cellular protein (VA cells), and 54 ng/mg cellular protein (V cells), within 48 h). Biological activity of these high concentrations of interleukin-6 measured in the enzyme-linked immuno-sorbent assay was confirmed in the B9-bioassay for interleukin-6 and by stimulation of alpha 2-macroglobulin production in rat liver parenchymal cell cultures. Lipopolysaccharide, interleukin-1 beta and tumor necrosis factor-alpha were potent stimulators of basal interleukin-6 production by VA and V cells, 1 microgram/ml lipopolysaccharide enhanced basal interleukin-6 production 3-fold within 48 h. 100 U/ml interleukin-1 beta and 1000 U/ml tumor necrosis factor-alpha each stimulated basal interleukin-6 production by VA cells 2-5 fold, whereas V cells were stimulated 10-25 fold. These effects were specific since the stimulation by lipopolysaccharide was completely inhibited by polymyxin B and the enhancing effects of interleukin-1 beta and tumor necrosis factor-alpha were neutralized by specific antibodies. Transforming growth factor-beta and interferon gamma did not influence interleukin-6 synthesis by either cell type in culture.

CONCLUSIONS

These results indicate that transformed fat-storing cells (VA cells) and fibroblasts (V cells) may function as a local source of interleukin-6 in the human liver. Since interleukin-6 plays a key role in the regulation of the production of acute phase proteins by liver parenchymal cells, we hypothesize that human liver (myo)fibroblasts may stimulate local production of acute phase proteins in the fibrotic liver, thus contributing to local regulation of inflammatory and fibrogenic reactions.

Interleukin 1 (IL1) increased phosphorylation of the small heat-shock protein (hsp 27) in MRC5 <em>fibroblasts</em>. The increase was maintained for at least 30 min, but levels had returned to pre-stimulation values by 2 h. When hsp 27 was metabolically labelled with [3H]leucine, about <em>15</em>% was phosphorylated in resting confluent cells; this rose to 90% upon stimulation by IL1. Peptide maps of the three differently charged phosphorylated forms were consistent with their arising by phosphorylation of increasing numbers of serine residues. IL1 had the same effect on hsp 27 in pig articular chondrocytes, endothelial cells from human umbilical vein and an epidermoid carcinoma cell line (KB). Certain other agents were found selectively to increase phosphorylation of hsp 27 in MRC5 cells besides IL1 [and tumour necrosis <em>factor</em> (TNF)]. Platelet-derived <em>growth</em> <em>factor</em> had a similar effect to that of IL1; bradykinin, acid <em>fibroblast</em> <em>growth</em> <em>factor</em> and ATP caused an intermediate effect; phorbol myristate acetate (PMA) and 1-oleoyl-2-acetylglycerol had smaller effects. Dibutyryl cyclic AMP and forskolin had no effects on hsp 27 phosphorylation. When cells had been depleted of protein kinase C (PKC) by prolonged treatment with PMA, stimulation by IL1, TNF or bradykinin still increased hsp 27 phosphorylation. The stimulation by all three agents was also unaffected by the PKC inhibitor staurosporine. IL1, TNF and bradykinin each caused hsp 27 phosphorylation by a pathway independent of PKC. The results are consistent with IL1 activating a serine kinase which remains to be identified.

Angiotensin II has been identified immunohistochemically in the ovaries of both rats and humans. Here we present evidence that angiotensin II (an extremely vasoactive agent in a wide range of tissues) may be involved in the regulation of the major steroidogenic enzyme in the ovary, cholesterol side chain cleavage cytochrome P-450 (P-450scc), as well as of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), which has been implicated as an angiogenic <em>factor</em> in the bovine corpus luteum. We have used primary cultures of bovine luteal cells to examine the effect of angiotensin II and its receptor antagonist, saralasin, on expression of mRNA encoding bFGF as well as on progesterone production and the expression of mRNA encoding cholesterol side chain cleavage cytochrome P-450 (P-450scc). Neither angiotensin II nor saralasin when added alone to the culture medium had any effect on basal progesterone production. Luteinizing hormone (LH) caused a <em>15</em>-fold increase in progesterone accumulation after 24 h of exposure which was reduced to 5-fold in the presence of angiotensin II. This appeared to be receptor-mediated in that although saralasin alone had no effect on LH-stimulated progesterone accumulation, it significantly reversed the inhibition by angiotensin II. This pattern was mirrored by the levels of mRNA encoding P-450scc, i.c., LH induced the highest levels of expression of this message, these levels were reduced by angiotensin II, and saralasin partially overcame this reduction. Levels of mRNA encoding bFGF were elevated by both LH and angiotensin II. Treatment with saralasin, however, resulted in complete inhibition of bFGF mRNA expression in the presence of both LH and angiotensin II. These results suggest a role for angiotensin II to mediate the action of LH as a regulator of bFGF expression and hence, potentially, angiogenesis. Local production of angiotensin II might also contribute to the refractoriness of luteal progesterone secretion to LH at the time of luteal regression.

During gestation, placental blood flow, endothelial nitric oxide (NO) production, and endothelial cell nitric oxide synthase (eNOS) expression are elevated dramatically. Shear stress can induce flow-mediated vasodilation, endothelial NO production, and eNOS expression. Both the activity and expression of eNOS are closely regulated because it is the rate-limiting enzyme essential for NO synthesis. The authors adapted CELLMAX artificial capillary modules to study the effects of pulsatile flow/shear stress on ovine fetoplacental artery endothelial (OFPAE) cell NO production, eNOS expression, and eNOS phosphorylation. This model allows for the adaptation of endothelial cells to low physiological flow environments and thus prolonged shear stresses. The cells were grown to confluence at 3 dynes/cm2, then were exposed to 10, <em>15</em>, or 25 dynes/cm2 for up to 24 h and NO production, eNOS mRNA, and eNOS protein expression were elevated by shear stress in a graded fashion (p < .05). Production of NO by OFPAE cells exposed to pulsatile shear stress was de novo; i.e., inhibited by L-NMMA (N(G)-monomethyl-L-arginine) and reversed by excess NOS substrate L-arginine. Rises in NO production at 25 dynes/cm2 (8-fold) exceeded (p < .05) that seen for eNOS protein (3.6-fold) or eNOS mRNA (1.5-fold). Acute rises in NO production with shear stress occurred by eNOS activation, whereas prolonged NO rises were via elevations in both eNOS expression and enzyme activation. The authors therefore used Western analysis to investigate the signaling mechanisms underlying pulsatile shear stress-induced increases in eNOS phosphorylation and protein expression by "flow-adapted" OFPAE cells. Increasing shear stress from 3 to <em>15</em> dynes/cm2 very rapidly increased eNOS Ser1177, ERK1/2 (extracellular signal-regulated kinase 1 and 2) and Akt, but not p38 MAPK (p38 mitogen-activated protein kinase) phosphorylation by Western analysis. Phosphorylation of eNOS Ser1177 under shear stress was elevated by 20 min, a response that was blocked by PI-3K (phosphatidylinositol 3-kinase) inhibitors wortmannin and LY294002, but not the MEK (MAPK kinase) inhibitor UO126. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) enhanced eNOS protein levels in static culture via a MEK-mediated mechanism, but it could not further augment the elevated eNOS protein levels induced by <em>15</em> dynes/cm2 shear stress. Blocking of either signaling pathways or p38 MAPK did not change the shear stress-induced increase in eNOS protein levels. Therefore, shear stress induced rapid eNOS phosphorylation on Ser1177 in OFPAE cells through a PI-3K-dependent pathway. The bFGF-induced rise in eNOS protein levels in static culture was much less than those observed under flow and was blocked by inhibiting MEK. Prolonged shear stress-stimulated increases in eNOS protein levels were not affected by inhibition of MEK- or PI-3K-mediated pathways. In conclusion, pulsatile shear stress greatly induces NO production by OFPAE cells through the mechanisms of both PI-3K-mediated eNOS activation and elevations in eNOS protein levels; bFGF does not further stimulate eNOS expression under flow condition.

The enzymatic pathways for formation of 1,2-diradylglyceride in response to epidermal <em>growth</em> <em>factor</em> in human dermal <em>fibroblasts</em> have been investigated. 1,2-Diradylglyceride mass was elevated 2-fold within one minute of addition of EGF. Maximal accumulation (4-fold) occurred at 5 minutes. Since both diacyl and ether-linked diglyceride species occur naturally and may accumulate following agonist activation, we developed a novel method to determine separately the alterations in diacyl and ether-linked diglycerides following stimulation of <em>fibroblasts</em> with EGF. Utilizing this method, it was found that approximately 80% of the total cellular 1,2-diradylglyceride was diacyl, the remaining 20% being ether-linked. Addition of EGF caused accumulation of 1,2-diacylglyceride without alteration in the level of ether-linked diglyceride. Thus, the observed induction of 1,2-diradylglyceride by EGF was due exclusively to increased formation of 1,2-diacylglyceride. In cells labelled with [3H]choline, the water soluble phosphatidylcholine hydrolysis products, phosphorylcholine and choline, were increased 2-fold within 5 minutes of addition of EGF. No hydrolysis of phosphatidylethanolamine, phosphatidylserine, or phosphatidylinositol was observed. Quantitation by radiolabel and mass revealed equivalent elevations in phosphorylcholine and choline, suggesting stimulation of both phospholipase C and phospholipase D activities. To identify the presence of EGF-induced phospholipase D activity, cells were labelled with exogenous [3H]1-0-hexadecyl, 2-acyl phosphatidylcholine and its conversion to phosphatidic acid in response to EGF determined. Radiolabelled phosphatidic acid was detectable in <em>15</em> seconds after addition of EGF and was maximal (3-fold) at 30 seconds. Consistent with the presence of EGF-induced phospholipase D activity, treatment of cells with EGF, in the presence of [14C]ethanol, resulted in the rapid formation of [14C]phosphatidylethanol, the product of phospholipase D-catalyzed transphosphatidylation. The formation of phosphatidylethanol, which competes for the formation of phosphatidic acid by phospholipase D, did not diminish the induction of 1,2-diglyceride by EGF. These data suggest that the phosphatidic acid formed by phospholipase D-catalyzed hydrolysis of phosphatidylcholine is not a major precursor of the observed increased 1,2-diglyceride. Thus, the induction of 1,2-diacylglycerol by EGF may occur primarily via phospholipase C-catalyzed hydrolysis of phosphatidylcholine.

An important feature of chronic obstructive pulmonary disease (COPD) is airway remodelling, the molecular mechanisms of which are poorly understood. In this study, the role of <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGF-1 and FGF-2) and their receptor, FGFR-1, was assessed in bronchial airway wall remodelling in patients with COPD (FEV1 < 75%; n = <em>15</em>) and without COPD (FEV1>> 85%; n = 16). FGF-1 and FGFR-1 were immunolocalized in bronchial epithelium, airway smooth muscle (ASM), submucosal glandular epithelium, and vascular smooth muscle. Quantitative digital image analysis revealed increased cytoplasmic expression of FGF-2 in bronchial epithelium (0.35 +/- 0.03 vs 0.20 +/- 0.04, p < 0.008) and nuclear localization in ASM (p < 0.0001) in COPD patients compared with controls. Elevated levels of FGFR-1 in ASM (p < 0.005) and of FGF-1 (p < 0.04) and FGFR-1 (p < 0.001) in bronchial epithelium were observed. In cultured human ASM cells, FGF-1 and/or FGF-2 (10 ng/ml) induced cellular proliferation, as shown by [3H]thymidine incorporation and by cell number counts. Steady-state mRNA levels of FGFR-1 were elevated in human ASM cells treated with either FGF-1 or FGF-2. The increased bronchial expression of <em>fibroblast</em> <em>growth</em> <em>factors</em> and their receptor in patients with COPD, and the mitogenic response of human ASM cells to FGFs in vitro suggest a potential role for the FGF/FGFR-1 system in the remodelling of bronchial airways in COPD.

Loss of heterozygosity at 10q26 was mapped using microsatellite markers in 20 osteosarcomas. A four-megabase region centered on marker D10S587 was affected by allelic loss in 60 percent of osteosarcomas. The most frequently lost marker was D10S1723. Around <em>15</em> known genes are found in this region. The gene immediately adjacent to D10S1723 encodes BUB3, an element of the spindle assembly mitotic checkpoint. Loss of BUB3 function could contribute to chromosomal instability. The <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2 (FGFR2) gene is located 2 Mb from the BUB3 gene and has the potential for a role in cancer. Inherited mutations of the FGFR2 gene result in skeletal dysplasias. FGFR2 alterations have also been implicated in gastric cancer. Human genome project data were used to design primers for amplifying FGFR2 in 18 genomic segments and BUB3 in 7 genomic segments. In each case, the segments encompassed coding exons and flanking intron sequences. The primers were used to search for mutations by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP). Several shifted bands were detected in the BUB3 exon 3 fragment. Sequencing resolved the BUB3 exon 3 fragment shifts into polymorphisms in intron 2. No mutations of BUB3 or FGFR2 were detected. It remains possible that BUB3 or FGFR2 hemizygosity alone contributes to osteosarcoma, or that one of the genes is cryptically inactivated by a higher-order modification or mutation outside the coding region. There may also be a yet undiscovered tumor suppressor gene in this region.

Identifying and understanding the early molecular events that underscore mineral pathogenicity using in vitro screening tests is imperative, especially given the large number of synthetic and natural fibers and particles being introduced into the environment. The purpose of the work described here was to examine the ability of gene profiling (Affymetrix microarrays) to predict the pathogenicity of various materials in a human mesothelial cell line (LP9/TERT-1) exposed to equal surface area concentrations (<em>15</em> x 10(6) or 75 x 10(6) microm(2)/cm(2)) of crocidolite asbestos, nonfibrous talc, fine titanium dioxide (TiO(2)), or glass beads for 8 or 24 h. Since crocidolite asbestos caused the greatest number of alterations in gene expression, multiplex analysis (Bio-Plex) of proteins released from LP9/TERT-1 cells exposed to crocidolite asbestos was also assessed to reveal if this approach might also be explored in future assays comparing various mineral types. To verify that LP9/TERT-1 cells were more sensitive than other cell types to asbestos, human ovarian epithelial cells (IOSE) were also utilized in microarray studies. Upon assessing changes in gene expression via microarrays, principal component analysis (PCA) of these data was used to identify patterns of differential gene expression. PCA of microarray data confirmed that LP9/TERT-1 cells were more responsive than IOSE cells to crocidolite asbestos or nonfibrous talc, and that crocidolite asbestos elicited greater responses in both cell types when compared to nonfibrous talc, TiO(2), or glass beads. Bio-Plex analysis demonstrated that asbestos caused an increase in interleukin-13 (IL-13), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), granulocyte colony-stimulating <em>factor</em> (G-CSF), and vascular endothelial <em>growth</em> <em>factor</em> (VEGF). These responses were generally dose-dependent (bFGF and G-CSF only) and tumor necrosis <em>factor</em> (TNF)-alpha independent (except for G-CSF). Thus, microarray and Bio-Plex analyses are valuable in determining early molecular responses to fibers/particles and may directly contribute to understanding the etiology of diseases caused by them. The number and magnitude of changes in gene expression or "profiles" of secreted proteins may serve as valuable metrics for determining the potential pathogenicity of various mineral types. Hence, alterations in gene expression and cytokine/chemokine changes induced by crocidolite asbestos in LP9/TERT-1 cells may be indicative of its increased potential to cause mesothelioma in comparison to the other nonfibrous materials examined.