BACKGROUND

We have previously reported activating mutations of the gene coding for the fibroblast growth factor receptor 3 (FGFR3) in invasive cervical carcinoma. To further analyze the role of FGFR3 in cervical tumor progression, we extended our study to screen a total of 75 invasive tumors and 80 cervical intraepithelial neoplasias (40 low-grade and 40 high-grade lesions).

RESULTS

Using single strand conformation polymorphism (SSCP) followed by DNA sequencing, we found FGFR3 mutation (S249C in all cases) in 5% of invasive cervical carcinomas and no mutation in intraepithelial lesions. These results suggest that, unlike in bladder carcinoma, FGFR3 mutation does not or rarely occur in non invasive lesions. Compared to patients with wildtype FGFR3 tumor, patients with S249C FGFR3 mutated tumors were older (mean age 64 vs. 49.4 years, P = 0.02), and were more likely to be associated with a non-16/18 HPV type in their tumor. Gene expression analysis demonstrated that FGFR3 mutated tumors were associated with higher FGFR3b mRNA expression levels compared to wildtype FGFR3 tumors. Supervised analysis of Affymetrix expression data identified a significant number of genes specifically differentially expressed in tumors with respect to FGFR3 mutation status.

CONCLUSIONS

This study suggest that tumors with FGFR3 mutation appear to have distinctive clinical and biological characteristics that may help in defining a population of patients for FGFR3 mutation screening.

Apoptotic neuronal death is known to occur in the developing brain and in the mature brain of patients with ischemic and degenerative disorders. Although microglial cells are known to become activated in specific conditions, it has not been elucidated whether they enhance or prevent neuronal apoptosis. The present study was intended to observe how microglial cells are involved in neuronal death. When rat primary cortical neurons were incubated with a nitric oxide (NO) donor sodium nitroprusside (SNP; 300 microM) for 10 min, neuronal death occurred 12-<em>16</em> hr later. The NO-induced neuronal death was inhibited by cycloheximide, and the SNP-treated neurons were characterized by nuclear fragmentation and intact cell membrane under electron microscopy. Agarose gel electrophoresis demonstrated DNA fragmentation of the SNP-treated neurons. Thus, the NO-induced neuronal death appeared to be apoptosis. When neurons were cocultured with rat primary microglial cells, the SNP treatment failed to induce the neuronal death. Because microglia-conditioned medium also prevented apoptotic neuronal death, microglial cells were considered to secrete antiapoptotic <em>factors</em>. The microglia-conditioned medium rescued neurons even when they were added to neuronal cultures after the SNP treatment, implying that the <em>factors</em> acted on neurons in a manner other than scavenging NO. Interleukin-3, interleukin-6, macrophage colony-stimulating <em>factor</em>, and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, which are known to be secreted by microglial cells, were not effective in preventing NO-induced neuronal death. Among microglia-derived substances, tumor necrosis <em>factor</em> alpha and plasminogen, which are heat-labile proteins, inhibited neuronal apoptosis. The neuroprotective action of the microglia-conditioned medium, however, still remained, even after it was heated. These findings suggest that microglial cells protect neurons against NO-induced lethal damage by secreting heat-labile and heat-stable neuroprotective <em>factors</em> in vitro.

This study evaluates the long-term outcome of farmer's lung (FL), adding high-resolution computed tomograms (HRCT) to previously reported procedures and verifying whether bronchoalveolar lavage (BAL) fluid markers or substrates of fibrosis (hyaluronic acid, Type III procollagen, fibronectin, and <em>fibroblast</em> <em>growth</em> <em>factors</em>) (FF) predict outcome. A total of 33 subjects with a history of FL dating back at least 6 yr were evaluated with pulmonary function tests, chest x-ray (CXR), and HRCT. All subjects had an initial evaluation, which included a BAL, 6 yr before the current study. Subjects were then either in acute FL (n = 19) or in clinical remission despite continued contact (n = 14). In the current study, pulmonary function tests revealed an obstructive profile in 13 subjects, restrictive changes in 1, an isolated decrease in lung diffusion capacity in 3, and normal values in <em>16</em>. Chest radiographs (CXR) were normal in 22 subjects, abnormal with interstitial or reticulonodular changes in 6, and suggestive of emphysema in 5. HRCT revealed emphysema in 9 subjects; 3 had localized fibrotic changes, 2 a ground-glass pattern, and 19 were normal. There was a good correlation between the findings on pulmonary function tests and HRCT; however, CXR alone did not suggest the existence of emphysema in 4 subjects who had such findings on HRCT. No correlations were found between most outcome parameters and the level of the BAL FF measured 6 yr previously. We conclude that airflow obstruction with or without emphysema is an important long-term sequela of FL and that BAL FF do not predict outcome in this disease.

<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-15) 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, -<em>16</em>, and -20, belongs to the FGF9 family, whereas EGL-17, like vertebrate FGF8, -17, and -18, could be included in the FGF8 family.

BACKGROUND

Growth factors including basic fibroblast growth factor (bFGF) are expected to be useful tools for enhancing osteochondral repair. However, suitable carriers are required to deliver a growth factor to the injury site. We evaluated the effects of intraarticular injection of bFGF with hyaluronic acid (HA) on osteochondral repair and the potential carrier role of HA in this treatment.

METHODS

Osteochondral defect was created in the medial femoral condyle of rabbits and received single or weekly intraarticular injection of bFGF (1 or 10 microg) with or without HA. Prior to the administration, bFGF was incubated with HA or vehicle-saline for 24 h at 4 degrees C. Four weeks after the initial injection, the animals were killed and the defect was evaluated grossly (12-point scale) and histologically (16-point scale). The effect of single injection of bFGF (1 microg) with HA was also compared to that of the carrier known as gelatin microspheres (GM) incorporating bFGF.

RESULTS

Weekly-administered bFGF alone induced undesirable side effects such as inflammatory responses and osteophyte formation. However, weekly-administered 1 mug of bFGF with HA yielded significantly better osteochondral repair than each treatment alone in gross and histological examinations with minimal side effects (P < 0.05). Single administration of 1 microg bFGF with HA but not GM incorporating bFGF showed significantly better osteochondral repair comparing to the vehicle control (P < 0.05).

CONCLUSIONS

Low-dose bFGF with HA was effective for osteochondral repair in rabbits. The significant osteochondral reparative role of bFGF with HA comparing with GM incorporating bFGF might be explained by the potential carrier role of HA and possible synergistic action between these two agents. The combination of HA with bFGF significantly suppressed the side effects resulting from single use of bFGF.

BACKGROUND

Chronic allograft nephropathy (CAN) is the most frequent cause of chronic dysfunction and late loss of renal allografts. Epithelial mesenchymal transition (EMT) has been identified as responsible for the presence of activated interstitial fibroblasts (myofibroblasts) and transforming growth factor beta (TGF-beta)/Smad is the key signaling mediator. It has been proposed that the bone morphogenetic protein 7 (BMP-7) antagonist, Gremlin, could participate in EMT, as a downstream mediator of TGF-beta.

METHODS

We evaluated 33 renal allograft biopsies, 16 of which showed CAN, versus 17 controls. By in situ hybridization we studied the expression of TGF-beta and Gremlin mRNA. Gremlin, BMP-7, E-cadherin, and alpha-smooth muscle actin (alpha-SMA) proteins were evaluated by immunohistochemistry and Smad3 activation by Southwestern. In cultured human tubuloepithelial cells (HK2 cell line), Gremlin induction by TGF-beta was studied by confocal microscopy.

RESULTS

Among renal biopsies of transplanted patients with CAN, we detected up-regulation of TGF-beta in colocalization with Gremlin (RNA and protein), mainly in areas of tubulointerstitial fibrosis. In the same tubules, we observed decreased expression of E-cadherin and induction of vimentin and alpha-SMA. BMP-7 was significantly decreased in the CAN biopsies. In addition, HK2 stimulated with TGF-beta (1 ng/mL) induced Gremlin production at 72 hours.

CONCLUSIONS

We postulated that Gremlin is a downstream mediator of TGF-beta, suggesting a role for Gremlin in EMT observed in CAN.

Fibrosis is the response of heart and other organs to injuries. Excessive fibrosis can cause organ dysfunction or even failure. Transforming-<em>growth</em> <em>factor</em> (TGF)-beta is a cytokine that induces <em>fibroblast</em> proliferation and increases the synthesis of a number of extracellular matrix proteins including collagens. Decorin (DCN) is a natural antagonist of TGF-beta. In the current study, we investigated the potential antifibrotic effects of DCN gene delivery by a recombinant adeno-associated viral (rAAV) vector to inhibit cardiac fibrosis in old, spontaneously hypertensive rats (SHRs), which develop severe cardiac and kidney fibrosis if without intervention. The rAAV-DCN vector was injected (at a dose of 1 x 10(11) vector genomes) via the tail vein into 5-month-old male SHRs, resulting in persistent, stable expression of DCN (up to <em>16</em> weeks). rAAV-DCN treatment significantly reduced collagen content and fibrosis in the heart and attenuated cardiomyocyte hypertrophy. Hemodynamics data at <em>16</em> weeks showed that DCN gene delivery induced a significant increase in left ventricular end-systolic pressure and maximal-minimal rate of pressure increase (+/-dp/dt(max)), but a decrease in left ventricular end-diastolic pressure (p < 0.05), compared with those of control animals. The expression of TGF-beta and alpha-smooth muscle actin, and the phosphorylation levels of Smad2 and p38 MAPK, were markedly reduced by rAAV-DCN treatment as compared with the controls. Thus, these results suggest that rAAV-mediated DCN overexpression led to the inhibition of hypertension-induced cardiac fibrosis and hypertrophy and improved cardiac function, and therefore may have therapeutic potential for organ fibrosis.

Clara cell protein (CC-<em>16</em>, also designated CC-10) is synthesized by the bronchiolar epithelium and has been suggested as an inhibitor of phospholipase A2 (PLA2) activity. Therefore, CC-<em>16</em> is a candidate for controlling inflammatory events in the lung. Because CC-<em>16</em> amounts and function may be altered in fibrosing lung diseases in which bronchiolar injury has been reported, it was measured in alveolar fluids and sera. Secretory PLA2 activity in alveolar fluids and the influence of CC-<em>16</em> on platelet-derived <em>growth</em> <em>factor</em>-induced human <em>fibroblast</em> chemotaxis and cytosolic PLA2 activity were also explored. CC-<em>16</em> content was decreased in alveolar fluids from idiopathic pulmonary fibrosis (IPF: 1.3 +/- 0.1 mg/L) and bleomycin lung (1.1 +/- 0.2 versus 2.1 +/- 0.2 mg/L in controls, p < 0.05), whereas there was a three- to ninefold increase in secretory PLA2 activity (p < 0.05 versus controls). CC-<em>16</em> inhibited <em>fibroblast</em> chemotaxis in a dose-dependent manner (90% inhibition at 30 micrograms/ml CC-<em>16</em>). This inhibition was reversed by reducing CC-<em>16</em>. CC-<em>16</em> was also able to lower <em>fibroblast</em>ic cytosolic PLA2 activity by 50% in vitro. In summary, CC-<em>16</em> is able to inhibit <em>fibroblast</em> chemotaxis in vitro by mechanisms that may be related to a blockage of cytosolic PLA2 activity. It can be postulated that CC-<em>16</em> deficiency may contribute to <em>fibroblast</em> burden activity in fibrosing lung diseases.

Although much is known about the structure of ras-encoded proteins, little is known about how expression is regulated. In serum-stimulated murine <em>fibroblasts</em>, c-ras-Ha mRNA levels fluctuated with the <em>growth</em> state but not with the position in the cell cycle. Two types of <em>growth</em> <em>factors</em> regulated c-ras-Ha expression: insulin (IN) or insulinlike <em>growth</em> <em>factor</em> I, each apparently acting through its cognate receptor, and epidermal <em>growth</em> <em>factor</em> (EGF). In quiescent cells, IN or insulinlike <em>growth</em> <em>factor</em> I induced c-ras-Ha mRNA three- to fivefold within 4 h, but thereafter the mRNA declined. By contrast, EGF had little effect in 4 h but induced the mRNA after 4 to 6 h. When quiescent cells were given serum or IN and EGF simultaneously, c-ras-Ha mRNA rose steadily, beginning 1 to 2 h after stimulation, and reached a stable five- to sevenfold elevation in <em>16</em> h. Thus, c-ras-Ha gene expression was sequentially regulated by two <em>growth</em> <em>factors</em>, one of which (IN) does not induce expression of other <em>growth</em>-regulated protooncogenes. A transformed derivative cell line that does not require IN for G1 progression has lost early IN-dependent but not late serum-dependent regulation. The results support the possibility that c-ras-Ha and IN action are functionally linked.

OBJECTIVE

To investigate the clotting and fibrinolytic activities in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and to examine the role of thrombin in synovial hyperplasia.

METHODS

We measured the amounts of thrombin-antithrombin-III complex (TAT), antithrombin-III (AT-III), thrombin, plasminogen, alpha 2-plasmin inhibitor (alpha 2-PI), and plasmin-alpha 2-antiplasmin complex (PAP) in SF of 20 patients with RA and <em>16</em> patients with osteoarthritis (OA). The proliferative response of synovial <em>fibroblast</em>-like cells to thrombin was measured using [3H] thymidine incorporation. Expression of platelet derived <em>growth</em> <em>factors</em> (PDGF) in conditioned medium was analyzed using a Western blot method, and expression of the mRNA of PDGF and their receptors was analyzed by reverse transcription polymerase chain reaction.

RESULTS

The amounts of clotting factors (TAT, AT-III) and fibrinolytic factors (plasminogen, alpha 2-PI, and PAP) were significantly higher in the patients with RA than in patients with OA (p < 0.01). Moreover, SF thrombin concentrations of patients with RA correlated significantly with erythrocyte sedimentation rates (rs = 0.751, p < 0.01) and serum C-reactive protein concentrations (rs = 0.531, p < 0.05). Thrombin exhibits mitogenic activity toward synovial fibroblast-like cells in vitro, and this mitogenic activity is associated with an increase in the expression of mRNA of both PDGF-alpha receptor and PDGF-beta-receptor.

CONCLUSIONS

The high levels of thrombin activity in the SF of patients with RA and strong mitogenic activity of thrombin toward the synovial fibroblast-like cells suggest that thrombin plays an important role in the pathogenesis of RA.

Radiation-induced gastrointestinal toxicity is due in part to the killing of the clonogenic crypt cells and eventual depopulation of the villi. Keratinocyte <em>growth</em> <em>factor</em> (KGF), a member of the <em>fibroblast</em> <em>growth</em> <em>factor</em> family (FGF-7), has been shown to stimulate proliferation of cells along the murine digestive tract from the foregut to the colon. Using an in vivo microcolony assay, we found that 1.0 mg/kg KGF administered intravenously (i.v.) for 3 consecutive days (2 days before, 1 day before and 2 h after irradiation) increased the number of surviving crypts by a <em>factor</em> of 2.6, 2.7 and 2.4 in the duodenum, jejunum and ileum, respectively, after a single-dose whole-body irradiation (10-<em>16</em> Gy) (P < 0.001). Treatment of mice with KGF i.v. significantly increased the D0 of the radiation survival curves by 0.37, 0.22 and 0.36 Gy, leading to dose modification <em>factors</em> of 1.28, 1.<em>16</em> and 1.24 for duodenal, jejunal and ileal crypt cells, respectively. Similar results were obtained with KGF administered subcutaneously. Treatment with both KGF and stem cell <em>factor</em> (previously shown to enhance intestinal crypt survival after total-body irradiation) increased the number of surviving crypt cells after irradiation to levels similar to that in animals treated with KGF alone. Administration of KGF for 7 consecutive days (beginning 2 days prior to irradiation) increased the LD(50/10) from 5.50 Gy/day to 5.90 Gy/day (P = 0.05) for animals irradiated with five daily fractions to a local abdominal field. These results suggest that KGF may be of clinical value in reducing radiation toxicity to the intestine.

We examined possible roles of keratinocyte <em>growth</em> <em>factor</em> (KGF) and hepatocyte <em>growth</em> <em>factor</em> (HGF) in lung morphogenesis. By polymerase chain reaction, transcripts for both KGF and its receptor were detected early (rat gestational days <em>16</em> and 14, respectively) and their abundance increased during lung morphogenesis. To evaluate possible role of KGF in lung morphogenesis, day 14 lung explants were cultured in Dulbecco's modified Eagle medium + 10% fetal calf serum for 1 to 4 days in the presence (5-50 ng/ml) or absence of KGF (control). KGF (at 25 and 50 ng/ml) induced a marked reduction in the number of terminal branches and destination of the distal epithelium into cyst-like structures. These effects of exogenous KGF were progressively diminished by increasing concentrations of anti-KGF (2-<em>16</em> micrograms/ml). Electron microscopic examination revealed that the epithelial cells of the cystic structures contained lamellar bodies, and were therefore type II cells and/or their progenitors. Northern blot analysis showed higher expression of surfactant protein C (SP-C) mRNA (a marker for alveolar epithelial type II cells) in KGF-treated fetal lungs. In situ hybridization of the KGF-treated lungs revealed that the SP-C mRNA-expressing cells were arranged distally in the form of linear arrays, a pattern distinctly different from that in control lungs. Acidic <em>fibroblast</em> <em>growth</em> <em>factor</em>, which also binds KGF receptors, in the presence of heparin mimicked the effect of KGF on branching. Transforming <em>growth</em> <em>factor</em>-beta(1) (TGF-beta 1) inhibited branching of fetal lungs in culture, and this effect dominated over that induced by KGF. Blocking of endogenous HGF with antibodies or addition of HGF to cultures of fetal lung explants had no significant effect on branching or <em>growth</em>. In conclusion, KGF markedly influences branching, and epithelial <em>growth</em>, differentiation, and patterning during lung morphogenesis.

OBJECTIVE

We investigated the effects of photobiomodulation (PBM) on cutaneous wound healing in an animal model of type II diabetes, Psammomys obesus (Sand Rats).

BACKGROUND

632-nm light has been established as the most effective wavelength for treatment of cutaneous wounds; however, the inconsistent efficacy of PBM may be due to inadequate treatment parameter selection.

METHODS

Using 632-nm light, an initial series of experiments were done to establish optimal treatment parameters for this model. Following creation of bilateral full-thickness skin wounds, non-diabetic Sand Rats were treated with PBM of differing dosages. Wound healing was assessed according to wound closure and histological characteristics of healing. Optimal treatment parameters were then used to treat type II diabetic Sand Rats while a diabetic control group received no irradiation. In order to elucidate the mechanism behind an improvement in wound healing, expression of basic fibroblast growth factor (bFGF) was assessed.

RESULTS

Significant improvement in wound healing histology and wound closure were found following treatment with 4 J/cm(2) (16 mW, 250-sec treatments for 4 consecutive days; p < 0.05). The 4 J/cm(2) dosage significantly improved histology and closure of wounds in the diabetic group in comparison to the non-irradiated diabetic group. Quantitative analysis of bFGF expression at 36 h post-injury revealed a threefold increase in the diabetic and non-diabetic Sand Rats after PBM.

CONCLUSIONS

The results demonstrate that PBM at an energy density of 4 J/cm(2) is effective in improving the healing of cutaneous wounds in an animal model of type II diabetes, suggesting that PBM (632 nm, 4 J/cm(2)) would be effective in treating chronic cutaneous wounds in diabetic patients.

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, 15, 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 <em>16</em>, 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.

Cytokine <em>growth</em> <em>factor</em> treatment of chronic wounds has met with mixed results. The chronic wound presents a hostile environment to peptides such as <em>growth</em> <em>factors</em>. Cytokine <em>growth</em> <em>factors</em> have not been studied extensively in acute wounds. However, incisional hernias are a major example of acute wound failure that has not been solved by various mechanical approaches. A biological approach to acute wound failure by use of cytokine <em>growth</em> <em>factors</em> may offer a new strategy. A rodent incisional hernia model was used. Seventy-six rats underwent 3-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 2-10 was infiltrated with 100 microl of vehicle alone or vehicle containing various test cytokine <em>growth</em> <em>factors</em>. Necropsy was performed on postoperative day 28 and the wounds were examined for herniation. Incisional hernias developed in 83 percent (13/<em>16</em>) of untreated incisional and 88 percent (7/8) and 83 percent (5/6) of the two vehicle-treated incisions (PBS and carboxymethylcellulose). Hernia incidences were decreased by priming of the fascial incision with transforming <em>growth</em> <em>factor</em>-beta(2) (12%, 1/8), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (25%, 2/8) and interleukin-1 beta (50%, 3/6) (p < 0.05). Aqueous platelet-derived <em>growth</em> <em>factor</em>, becaplermin, insulin-like <em>growth</em> <em>factor</em>, and granulocyte macrophage-colony stimulating <em>factor</em> did not significantly decrease the incidence of acute wound failure (p>> 0.05). A biological approach to acute wound failure as measured by incisional hernia formation can be useful in reducing the incidence of this complication. Transforming <em>growth</em> <em>factor</em>-beta(2), basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and interleukin 1 beta all eliminated or significantly reduced the development of incisional hernias in the rat model.

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 = 15) and without COPD (FEV1>> 85%; n = <em>16</em>). 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.

The angiogenic proteins basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF; FGF-2) and vascular endothelial <em>growth</em> <em>factor</em> 121 (VEGF(121)) are each able to enhance the collateral-dependent blood flow after bilateral femoral artery ligation in rats. To study the effect of nitric oxide (NO) synthase (NOS) inhibition on bFGF- or VEGF(121)-induced blood flow expansion, the femoral arteries of male Sprague-Dawley rats were ligated bilaterally, and the animals were given tap water [non-N(G)-nitro-L-arginine methyl ester (L-NAME) group; n = 36] or water that contained L-NAME (L-NAME group; 2 mg/ml, n = 36). Animals from each group were further divided into three subgroups: vehicle (n = 12), bFGF (5 microg x kg(-1) x day(-1), n = 12), or VEGF(121) (10 microg x kg(-1) x day(-1), n = 12). <em>Growth</em> <em>factors</em> were delivered via intra-arterial infusion with osmotic pumps over days 1-14. On day <em>16</em>, after a 2-day delay to permit clearance of bFGF and VEGF from the circulation, maximal collateral blood flow was determined by (85)Sr- and (141)Ce-labeled microspheres during treadmill running. L-NAME (approximately 137 mg x kg(-1) x day(-1)) for 18 days increased systemic blood pressure (approximately 26%, P<0.001). In the absence of L-NAME, collateral-dependent blood flows to the calf muscles were greater in the VEGF(121)- and bFGF-treated subgroups (85 +/- 4.5 and 80 +/- 2.9 ml x min(-1) x 100 g(-1), respectively) than in the vehicle subgroup (49 +/- 3.0 ml x min(-1) x 100 g(-1), P<0.001). In the presence of NOS inhibition by L-NAME, blood flows to the calf muscles were essentially equivalent among the three subgroups (54 +/- 3.0, 56 +/- 5.1, and 47 +/- 2.0 ml x min(-1) x 100 g(-1) in the bFGF-, VEGF(121)-, and vehicle-treated subgroups, respectively) and were not different from the blood flow in the non-L-NAME vehicle subgroup. Our results therefore indicate that normal NO production is essential for the enhanced vascular remodeling induced by exogenous bFGF or VEGF(121) in this rat model of experimental peripheral arterial insufficiency. These results imply that a blunted endothelial NO production could temper vascular remodeling in response to these angiogenic <em>growth</em> <em>factors</em>.

Eleven athymic nude rats had stereotactic intracerebral inoculation of cells from one of three established human glioma cell lines (A172, A1207, and A1235). The implants grew progressively in 9 of 11 instances, which led to spontaneous death of the host in 14 to 37 days. For comparison, two Sprague-Dawley normal albino rats were implanted with the rat C6 glioma cell line. One rat died at 14 days, and the other was killed at Day <em>16</em>. The human glioma cells developed into partially (A172, A1235) or totally (A1207) circumscribed tumor masses. Invasion, when present, was manifested as infiltrating prongs of cells rather than as individual cell infiltration. The <em>growth</em> of the human glioma cells was accompanied by a small zone of surrounding edema and marked central necrosis. These features were not encountered in the C6 implants. Inflammatory changes were minimal to nonexistent in all cases. All tumor lines produced internal cerebral herniation and neuraxis spread with implants seeded throughout the ventricular system, often associated with ventricular dilation. In situ hybridization, by the use of isotopic and nonisotopic detection methods, was used to study the cellular expression of the acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> genes in A172 glioma xenografts. The expression of these genes was not seen in normal rat brain, but the genes were selectively overexpressed by the glioma cell implants, with especially high signal in the tumor periphery.(ABSTRACT TRUNCATED AT 250 WORDS)

Dysregulation of miRNAs has been shown to contribute to the carcinogenesis and progression of nasopharyngeal carcinoma (NPC). Our previous microarray data showed that miR-<em>16</em> expression is significantly decreased in archived NPC tissues. Here, we confirmed that miR-<em>16</em> was reduced in NPC cell lines and freshly-frozen samples. Ectopic expression of miR-<em>16</em> suppressed NPC cell proliferation, migration, and invasion in vitro and inhibited tumor <em>growth</em> and metastatic colonization in the lung in vivo. Furthermore, <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2) was identified as a direct target of miR-<em>16</em>, and both phosphoinositide-3- kinase/AKT (PI3K/AKT) and mitogen-activated protein kinase (MAPK) signaling pathways were repressed after miR-<em>16</em> overexpression. In addition, the restoration of FGF2 reversed the suppressive effects of miR-<em>16</em>. Together, these results indicated that miR-<em>16</em> suppresses NPC carcinogenesis and progression by targeting FGF2, thereby representing a potential target for miRNA-based therapy for NPC in the future.

The serum level and urinary output of basic and acidic <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) were measured by sandwich enzyme immunoassay (EIA) in patients with renal cell carcinoma. In over fifty percent (<em>16</em>/31) of renal cell carcinoma patients, basic FGF was elevated (greater than 30 pg/ml) in their sera. There is relatively good correlation between serum levels of basic FGF and tumor stage or grade, while urinary daily output of basic FGF did not correlate with increased malignancy. The present results indicate that serum basic FGF level of patients with renal cell carcinoma is a useful diagnostic and prognostic marker for renal cell carcinoma. On the other hand, acidic FGF was not detectable in all sera and urine.

OBJECTIVE

The cornea is an avascular organ, where induction of new blood vessels involves the turn-on of proangiogenic <em>factors</em> and/or the turn-off of antiangiogenic regulators. Prolactin (PRL) fragments of 14 kDa and <em>16</em> kDa bind to endothelial cell receptors and inhibit angiogenesis. This study was designed to determine whether antiangiogenic PRL-like molecules are involved in cornea avascularity.

METHODS

Sixteen-kDa PRL and basic fibroblast growth factor (bFGF) or anti-PRL antibodies were placed into rat cornea micropockets and neovascularization evaluated by the optical density associated with capillaries stained by the peroxidase reaction and by the number of vessels growing into the implants. Prolactin receptors in corneal epithelium were investigated by immunocytochemistry.

RESULTS

bFGF induced a dose-dependent stimulation of corneal neovascularization. This effect was inhibited by coadministration of <em>16</em>-kDa PRL, as indicated by a 65% reduction in vessel density and a 50% decrement in the incidence of angiogenic responses. Corneal angiogenic reactions of different intensities were induced by implantation of polyclonal and monoclonal anti-PRL antibodies. Corneal epithelial cells were labeled by several anti-PRL receptor monoclonal antibodies.

CONCLUSIONS

These findings show that exogenous <em>16</em>-kDa PRL inhibits bFGF-induced corneal neovascularization and suggest that PRL-like molecules with antiangiogenic actions function in the cornea. PRL receptors in the corneal epithelium may imply that PRL in the cornea derives from lacrimal PRL internalized through an intracellular pathway. These observations are consistent with the notion that members of the PRL family are potential regulators of corneal angiogenesis.

The E5 proteins of bovine papillomavirus type 1 (BPV-1) and human papillomavirus type <em>16</em> (HPV-<em>16</em>) are small (44-83 amino acids), hydrophobic polypeptides that localize to membranes of the Golgi apparatus and endoplasmic reticulum, respectively. While the oncogenic properties of BPV-1 E5 have been characterized in detail, less is known about HPV-<em>16</em> E5 due to its low expression in mammalian cells. Using codon-optimized HPV-<em>16</em> E5 DNA, we have generated stable <em>fibroblast</em> cell lines that express equivalent levels of epitope-tagged BPV-1 and HPV-<em>16</em> E5 proteins. In contrast to BPV-1 E5, HPV-<em>16</em> E5 does not activate <em>growth</em> <em>factor</em> receptors, phosphoinositide 3-kinase or c-Src, and fails to induce focus formation, although it does promote anchorage-independent <em>growth</em> in soft agar. These variant activities are apparently unrelated to differences in intracellular localization of the E5 proteins since retargeting HPV-<em>16</em> E5 to the Golgi apparatus does not induce focus formation.

BACKGROUND

It was previously demonstrated that the dipeptide carnosine inhibits growth of cultured cells isolated from patients with malignant glioma. In the present work we investigated whether carnosine also affects tumor growth in vivo and may therefore be considered for human cancer therapy.

RESULTS

A mouse model was used to investigate whether tumor growth in vivo can be inhibited by carnosine. Therefore, NIH3T3 fibroblasts, conditionally expressing the human epidermal growth factor receptor 2 (HER2/neu), were implanted into the dorsal skin of nude mice, and tumor growth in treated animals was compared to control mice. In two independent experiments nude mice that received tumor cells received a daily intra peritoneal injection of 500 microl of 1 M carnosine solution. Measurable tumors were detected 12 days after injection. Aggressive tumor growth in control animals, that received a daily intra peritoneal injection of NaCl solution started at day 16 whereas aggressive growth in mice treated with carnosine was delayed, starting around day 19. A significant effect of carnosine on tumor growth was observed up to day 24. Although carnosine was not able to completely prevent tumor growth, a microscopic examination of tumors revealed that those from carnosine treated animals had a significant lower number of mitosis (p < 0.0003) than untreated animals, confirming that carnosine affects proliferation in vivo.

CONCLUSIONS

As a naturally occurring substance with a high potential to inhibit growth of malignant cells in vivo, carnosine should be considered as a potential anti-cancer drug. Further experiments should be performed in order to understand how carnosine acts at the molecular level.

The family of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptors (FGFRs) is encoded by four distinct genes. FGFR1 and FGFR4 are both expressed during myogenesis, but whereas the function of FGFR1 in myoblast proliferation has been documented, the role of FGFR4 remains unknown. Here, we report on a new splice form of FGFR4 cloned from primary cultures of mouse satellite cells. This form, named FGFR4(-<em>16</em>), lacks the entire exon <em>16</em>, resulting in a deletion within the FGFR kinase domain. Expression of FGFR4(-<em>16</em>) coincided with that of wild-type FGFR4 in all FGFR4-expressing tissues examined. Moreover, expression of both FGFR4 forms correlated with the onset of myogenic differentiation, as determined in mouse C2C12 cells and in the inducible myogenic system of 10T(1/2)-MyoD-ER cell line. Both endogenous and overexpressed forms of FGFR4 exhibited N-glycosylation. In contrast to FGFR1, induced homodimerization of FGFR4 proteins did not result in receptor tyrosine phosphorylation. Surprisingly, coexpression of FGFR4 forms and a chimeric FGFR1 protein resulted in FGFR4 tyrosine phosphorylation, raising the possibility that FGFR4 phosphorylation might be enabled by a heterologous tyrosine kinase activity. Collectively, the present study reveals novel characteristics of mouse FGFR4 gene products and delineates their expression pattern during myogenesis. Our findings suggest that FGFR4 functions in a distinctly different manner than the prototype FGFR during myogenic differentiation.