BACKGROUND

Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease of premature birth, characterized by impaired alveolar development and inflammation. Pathomechanisms contributing to BPD are poorly understood. However, it is assumed that genetic factors predispose to BPD and other pulmonary diseases of preterm neonates, such as neonatal respiratory distress syndrome (RDS). For association studies, genes upregulated during alveolarization are major candidates for genetic analysis, for example, matrix metalloproteinases (MMPs) and fibroblast growth factors (FGFs) and their receptors (FGFR).

OBJECTIVE

Determining genetic risk variants in a Caucasian population of premature neonates with BPD and RDS. Methods. We genotyped 27 polymorphisms within 14 candidate genes via restriction fragment length polymorphism (RFLP): MMP-1, -2, -9, and -12, -16, FGF receptors 2 and 4, FGF-2, -3, -4, -7, and -18, Signal-Regulatory Protein α (SIRPA) and Thyroid Transcription Factor-1 (TTF-1).

RESULTS

Five single nucleotide polymorphisms (SNPs) in MMP-9, MMP-12, FGFR-4, FGF-3, and FGF-7 are associated (P < 0.05) with RDS, defined as surfactant application within the first 24 hours after birth. One of them, in FGFR-4 (rs1966265), is associated with both RDS (P = 0.003) and BPD (P = 0.023).

CONCLUSIONS

rs1966265 in FGF receptor 4 is a possible genetic key variant in alveolar diseases of preterm newborns.

The sulfonic acid polymers poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS), poly(4-styrenesulfonic acid) (PSS), and poly(anetholesulfonic acid) (PAS) proved to be highly potent inhibitors of angiogenesis in the chick chorioallantoic membrane (CAM) assay. PAMPS was found to achieve a dose-dependent inhibition of microvessel formation in the CAM assay ranging from 57 +/- <em>16</em>% inhibition at 10 micrograms/disc to 72 +/- 15% at 150 micrograms/ disc. Also, PSS and PAS caused a strong inhibition of angiogenesis (55 +/- 19% and 48 +/- <em>16</em>%, respectively, at 50 micrograms/disc), whereas poly(vinylsulfonic acid) (PVS) was found to be inactive at this dose. The compounds proved to be nontoxic for the developing chick embryo at these doses. Suramin, which was included as a reference compound, caused only a slight inhibition of vascular density, at a dose of 150 micrograms/disc, whereas pentosan polysulfate (PPS) was found to be toxic. PAMPS, PAS, and PSS, but not PVS, inhibited microvessel formation in the rat aorta-ring assay. In addition, the increased [3H-methyl]dThd uptake in endothelial cells in vitro upon stimulation with basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) was inhibited by PAMPS, PAS, and PSS at 20 micrograms/ml. A strong correlation (r = 0.95) was found between the antiangiogenic effect of the sulfonic acid polymers in the CAM assay and their inhibition of the bFGF-induced mitogenic response, indicating that bFGF is the target for these sulfonic acid polymers. These results suggest that sulfonic acid polymers, and in particular PAMPS, may be considered as specific, nontoxic angiogenesis inhibitors.

OBJECTIVE

Photoreceptors can be prevented from undergoing apoptosis in response to constant light by the application of exogenous neuroprotective agents, including brain-derived neurotrophic factor (BDNF). BDNF, however, cannot exert its effect directly on photoreceptors because they do not express receptors for BDNF. It has been proposed that BDNF released from Müller cells provides a feed-forward loop, increasing ciliary neurotrophic factor (CNTF) and basic fibroblast growth factor (bFGF) production in Müller cells, which may enhance photoreceptor survival. The authors hypothesized that retinas with reduced BDNF levels in which the BDNF-mediated release of neuroprotective signals is dampened are more susceptible to light-induced photoreceptor degeneration.

METHODS

Young adult BDNF+/+ and BDNF+/- littermates (B6.129-BDNF(tm1-LT)) were analyzed. Retinal neurotrophin and growth factor mRNA levels were determined by quantitative RT-PCR, photoreceptor function was assessed through electroretinography, and survival was documented in morphologic sections and in TUNEL assays. Oxidative stress was assayed by measuring glutathione peroxidase activity.

RESULTS

At baseline, BDNF+/- animals had significantly increased levels of glial-derived neurotrophic factor (GDNF) mRNA compared with their wild-type littermates. After light damage GDNF, CNTF, and BDNF mRNA levels dropped 14- to 16-fold in the BDNF+/+ mice but remained almost unchanged compared with baseline levels in the BDNF+/- mice. Preservation of neurotrophin levels in BDNF+/- mice correlated with photoreceptor cell survival, preservation of function, and reduced oxidative stress.

CONCLUSIONS

Contrary to the hypothesis, reducing BDNF levels resulted in photoreceptor protection against light damage. Survival was paralleled by a reduction in oxidative stress and the preservation of neurotrophin levels, suggesting that chronic reduction of BDNF in the retina provides a level of preconditioning against stress.

Expression of epidermal <em>growth</em> <em>factor</em> (EGF) and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) was examined in 56 patients with hepatocellular carcinoma (HCC) using an immunohistochemical method. EGF and FGF were expressed on carcinoma cells in 14 (25%) and 23 cases (41%), respectively. In the 23 FGF-positive cases, 11 cases were positive for both acidic and basic FGF, while 18 were positive for acidic FGF, and <em>16</em> were positive for basic FGF. In non-cancerous hepatic tissues, FGF was weakly positive in macrophages, hepatocytes and vascular endothelial cells in some cases, while EGF was totally negative. There were no significant correlations between the expression of EGF or FGF on carcinoma cells and the various clinicopathologic <em>factors</em> examined. These data suggest that EGF and FGF are produced by human HCC cells in vivo. The roles of the expression of these <em>growth</em> <em>factors</em> in the development and progression of HCC remain only speculative.

OBJECTIVE

To study the processes involved in mediating conjunctival remodelling in vernal keratoconjunctivitis (VKC) by investigating the expression of integrin receptors, epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), transforming growth factor-beta(TGF-beta), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and Ki67 antigen, which is a marker for cell proliferation.

METHODS

Conjunctival biopsy specimens from 16 patients with active VKC and nine control subjects were studied by immunohistochemical techniques using monoclonal and polyclonal antibodies directed against the integrin alpha3 and alpha6 subunits, EGFR, VEGF, TGF-beta, bFGF, PDGF, and Ki67 antigen. The phenotype of inflammatory cells expressing growth factors was examined by double immunohistochemistry.

RESULTS

In the normal conjunctiva, very weak immunoreactivity was observed for EGFR and VEGF in epithelial cells, and for alpha3 and alpha6 integrin subunits on basal epithelial cells, and on vascular endothelial cells in the upper substantia propria. There was no immunoreactivity for the other antibodies. In VKC specimens, strong staining for alpha3 and alpha6 integrin subunits was observed on the membranes of basal and suprabasal epithelial cells, and all vascular endothelial cells. Immunoreactivity for Ki67 antigen was observed in the nuclei of the basal and suprabasal epithelial cells. Strong immunoreactivity was observed for EGFR in the deeper layers of the epithelium, and for VEGF in all epithelial cells. Inflammatory cells expressing EGFR, VEGF, TGF-beta, bFGF, and PDGF were noted in 8, 9, 11, 10, and 10 specimens, respectively. The majority of inflammatory cells expressing growth factors were eosinophils (45+/-4%) and monocytes/macrophages (35+/-4%).

CONCLUSIONS

Chronic conjunctival inflammation in VKC is associated with increased staining of alpha3, and alpha6 integrin subunits, EGFR, VEGF, TGF-beta, bFGF, and PDGF that might mediate conjunctival remodelling.

Three cell lines derived from fin (WSF), head soft tissue (WSHST) and body muscle (WSBM) were established from white sturgeon (Acipenser transmontanus). Characterization included determination of optimal <em>growth</em> kinetics, karyotyping, and mitochondrial ribosomal RNA (rRNA) genotyping. The primary cultures of these cells were generated by the explant technique using the L-15 medium supplemented with 20% fetal bovine serum and epidermal/<em>fibroblast</em> <em>growth</em> <em>factors</em>. The cells grew between 15-30 degrees C, but optimal <em>growth</em> occurred at 25 degrees C with a doubling time of 48 hours. The cell lines can be readily maintained in-vitro and have been subcultured over 35 times. Following cryopreservation in liquid nitrogen, thawed cells exhibit a viability of>> 90% after a <em>16</em>-month storage period. Chromosomal typing of these cell lines at their 17th passage revealed a chromosomal distribution of 242 to 278 with an apparent peak ranging from 250 to 260. Polymerase chain reaction amplification of mitochondrial <em>16S</em> ribosomal RNA and sequence analysis indicated 100% identity of the sequences found in the cell lines with those found in the source animal, confirming that the cell lines were of A. transmontanus origin.

An aberrant elevation in intraneuronal calcium levels resulting from energy failure and excitatory amino acid receptor activation is believed to play a major role in the neuronal damage and death that occur in stroke. We have found that several <em>growth</em> <em>factors</em> can protect cultured rat hippocampal and septal neurons and human cortical neurons from excitotoxic damage caused by glucose deprivation or hypoxia. Using the calcium indicator dye fura 2 and whole-cell patch-clamp recording, we found that glucose deprivation initially results in calcium current inhibition and a reduction in intraneuronal free calcium levels without morphological signs of cell damage. After 12 to <em>16</em> hours of glucose deprivation, a large elevation in intraneuronal calcium levels occurred that involved N-methyl-D-aspartate receptor activation and mediated the cell damage and death. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), nerve <em>growth</em> <em>factor</em> (NGF), and insulin-like <em>growth</em> <em>factors</em> (IGF-I and IGF-II) each prevented, in a dose-dependent manner, glucose deprivation-induced loss of calcium homeostasis and neuronal damage. The <em>growth</em> <em>factors</em> were effective to varying degrees when added up to 12 hours after the onset of glucose deprivation. NGF, bFGF, and IGFs also protected neurons against damage caused by exposure to a hypoxic environment. By stabilizing intraneuronal calcium levels within a window of concentrations conducive to neuronal survival, <em>growth</em> <em>factors</em> can protect neurons against the damaging effects of ischemia-like insults. Because ATP levels are expected to be reduced under ischemia-like conditions, we determined whether the <em>growth</em> <em>factors</em> would protect neurons against a more selective reduction in ATP levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-6 (IL-6) is a pleiotropic mediator of immune function and a <em>growth</em> <em>factor</em> for a variety of hematopoietic cell types. Because IL-1 beta is known to induce IL-6 production in nonplacental mesenchymal cells and is locally produced by maternal decidua, this study was designed to determine whether IL-1 beta could regulate IL-6 production by second trimester placental villous core mesenchymal cells (VCMC) in vitro. VCMC were prepared for culture by enzymatic digestion of placentas (14-20 weeks gestation; n = 7). Immunohistochemistry performed on the confluent cells demonstrated that more than 95% of the cells had a <em>fibroblast</em>-like morphology and were vimentin positive, less than 5% were leukocyte common antigen (CA-45) positive, and no trophoblast contamination was demonstrated by the lack of cytokeratin staining. In dose-response experiments, a specific dose-response induction of IL-6 mRNA expression and IL-6-immunoreactive protein production by IL-1 beta was demonstrated; this was first seen at 100 pg/ml IL-1 beta [455 +/- 191 ng/ml (+/- SEM); controls, 42 +/- <em>16</em> ng/ml; P < 0.05]. In time-course studies, the addition of 10 ng/ml IL-1 beta significantly increased IL-6 production rates; this was first seen at 8 h of culture and increased in a linear fashion up to 48 h. At 48 h of culture, IL-6 levels were 17 times higher in treated VCMC (861 +/- 179 ng/ml) compared to those in nontreated VCMC (51 +/- 14 ng/ml). In summary, IL-1 beta stimulates VCMC IL-6 production in a specific dose- and time-dependent manner. From these results, we conclude that VCMC are an important source of IL-6 in second trimester placenta and that production of placental IL-6 be may regulated by decidual IL-1 beta.

The IL-1R antagonist protein (IRAP) is a competitive inhibitor of IL-1, which is predominantly synthesized by monocytes. We show that this molecule is also expressed in human synovial <em>fibroblasts</em> and dermal <em>fibroblasts</em> (CRL 1445). IRAP mRNA was regulated in a time- and dose-dependent manner by IL-1 alpha, TNF-alpha, LPS, and PMA. Maximal induction of IRAP mRNA was observed between 8 and <em>16</em> h after stimulation with IL-1 alpha (1 U/ml), TNF-alpha (10 U/ml), LPS (50 ng/ml), and PMA (10 ng/ml). Their relative efficacy was as follows: PMA>> LPS>> IL-1 alpha>> TNF-alpha. Potentiation was observed when <em>fibroblasts</em> were treated with IL-1 alpha plus basic <em>fibroblast</em> <em>growth</em> <em>factor</em> and IL-1 alpha plus platelet-derived <em>growth</em> <em>factor</em>-BB homodimer. Although LPS and PMA were the best inducers of IRAP mRNA, quantitation of the IRAP protein revealed that its synthesis and release were differentially regulated. Immunoprecipitation and SDS-PAGE of culture supernatant from LPS-treated cells and cell lysates of <em>fibroblasts</em> treated with LPS or PMA showed a single IRAP band with a molecular mass of approximately 22 kDa. Very little IRAP was detected in culture supernatants of cells treated with PMA. Quantitation of IRAP revealed that LPS induced the synthesis of secreted IRAP that was released, whereas the majority of the protein induced by PMA remained cell-associated. Reverse transcriptase-polymerase chain reaction amplification demonstrated that although LPS and PMA induced both transcripts, LPS preferentially induced secreted IRAP, whereas PMA differentially induced intracellular IRAP mRNA. <em>Fibroblasts</em> synthesize at least two different forms of IRAP depending on the inducing signal, and may regulate the inflammatory response by dampening the proinflammatory effects of IL-1 via a negative feedback mechanism with IRAP. The relative importance of <em>fibroblast</em> sIRAP vs intracellular IRAP in regulating the inflammatory response by the connective tissue remains to be determined.

It is increasingly appreciated that the properties of a biomaterial used in intramyocardial injection therapy influence the outcomes of infarcted hearts that are treated. In this report the extended in vivo efficacy of a thermally responsive material that can deliver dual <em>growth</em> <em>factors</em> while providing a slow degradation time and high mechanical stiffness is examined. Copolymers consisting of N-isopropylacrylamide, 2-hydroxyethyl methacrylate, and degradable methacrylate polylactide were synthesized. The release of bioactive basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and insulin-like <em>growth</em> <em>factor</em> 1 (IGF1) from the gel and loaded poly(lactide-co-glycolide) microparticles was assessed. Hydrogel with or without loaded <em>growth</em> <em>factors</em> was injected into 2 week-old infarcts in Lewis rats and animals were followed for <em>16</em> weeks. The hydrogel released bioactive bFGF and IGF1 as shown by mitogenic effects on rat smooth muscle cells in vitro. Cardiac function and geometry were improved for <em>16</em> weeks after hydrogel injection compared to saline injection. Despite demonstrating that left ventricular levels of bFGF and IGF1 were elevated for two weeks after injection of <em>growth</em> <em>factor</em> loaded gels, both functional and histological assessment showed no added benefit to inclusion of these proteins. This result points to the complexity of designing appropriate materials for this application and suggests that the nature of the material alone, without exogenous <em>growth</em> <em>factors</em>, has a direct ability to influence cardiac remodeling.

The role of interstitial vs. alveolar macrophages in the generation of pulmonary fibrosis after silica was examined. Using whole body irradiation to delay the inflammatory response and so retard particulate clearance, many more instilled silica particles reached the interstitial macrophages in the first 2 weeks than after silica alone. This was followed by greatly increased <em>fibroblast</em> proliferation and deposition of collagen in the irradiation plus silica group, which developed large interstitial granulomas at the sites of silica retention. Although alveolar macrophages containing silica were seen in both silica groups, more interstitial particles were observed after combined irradiation and silica, significantly more silica was recovered in a residue from the lungs at <em>16</em> weeks, and pulmonary fibrosis at 8-<em>16</em> weeks was greater than in all other groups. The results indicate that increased <em>fibroblast</em> <em>growth</em> and collagen synthesis in vivo are associated with phagocytosis of silica by interstitial macrophages rather than by free alveolar macrophages. It is suggested that transfer of a macrophages-derived <em>growth</em> <em>factor</em> to <em>fibroblasts</em> is more efficient when it occurs within the pulmonary interstitium.

The <em>growth</em> kinetics of subcultured human synovial <em>fibroblasts</em> from <em>16</em> patients with inflammatory and noninflammatory arthropathies were studied in antibiotic free media. The experimental design allowed a clear distinction between the <em>growth</em> rates and final saturation densities achieved. The effects of refeeding and of the serum concentration were evaluated for each line. Inflammatory lines achieved significantly higher final saturation densities and <em>growth</em> rates than noninflammatory lines for most protocols, but the differences between rheumatoid and nonrheumatoid groups were less marked. Inflammaroty <em>fibroblasts</em> demonstrated a greater independence to nutritional and <em>growth</em> stimulatory <em>factors</em> in their microenvironment than noninflammatory <em>fibroblasts</em>.

BACKGROUND

Transforming growth factor-beta (TGF-beta) is composed of a family of multifunctional polypeptide growth factors involved in embryogenesis, inflammation, regulation of immune response, angiogenesis, wound healing, and extracellular matrix formation. TGF-beta1 is the most common isoform found in human tissues. A role of TGF-beta in the pathogenesis of periodontal disease has been suggested. The aim of the present study was a comparative immunohistochemical evaluation of TGF-beta1 in normal keratinized gingiva and in the peri-implant soft tissues surrounding failing non-submerged implants.

METHODS

Twenty patients participated in this study. Ten biopsies from healthy keratinized mucosa and 10 biopsies from peri-implant soft tissues surrounding failing implants were obtained (one biopsy per patient). The biopsies were obtained from different patients.

RESULTS

In 5 cases of healthy mucosa, the stromal cells were positive between 1 to 5. In 7 cases, the epithelial layers were positive, between 1 and 18 cells. The superficial epithelial layer was negative in all cases. In 9 cases, there was a positivity of the vascular component, between 2 and 16 vessels. In failing implants, the stromal cells were positive in 6 cases, between 1 and 4. In all cases, cells of the epithelial layers were positive, between 15 and 40. The vascular component was positive in all cases, between 12 and 30 vessels. The differences between TGF-beta1 expression in the epithelium around healthy and failing implants were statistically significant (P < 0.0001). The differences between TGF-beta1 expression in the blood vessels in the soft tissues around healthy and failing implants were also statistically significant (P < 0.0001). No statistically significant difference was observed between the 2 groups in the TGF-beta1 expression in the stromal cells (P = 0.88).

CONCLUSIONS

TGF-beta1 may be one of the most important factors in the regulation of the infiltrate, and in the production of tissue repair with a stimulation of fibroblasts and endothelial cells.

OBJECTIVE

Preeclampsia is characterized by maternal hypertension, proteinuria, edema, and shallow placental invasion. Insulin-like growth factor binding protein-1, abundant in maternal decidua, is believed to play a role in limiting trophoblast invasiveness. In this study we addressed the hypothesis that this binding protein is aberrantly expressed in preeclampsia. We also investigated circulating levels of insulin-like growth factor-I and insulin-like growth factor-II in subjects with severe preeclampsia compared with controls.

METHODS

Insulin-like growth factor binding protein-1 was investigated by immunohistochemistry at the maternal-fetal interface of eight pregnancies complicated by severe preeclampsia and six controls between 21 and 34 weeks of gestation. Cell types were identified with use of cell-specific markers. Circulating levels of insulin-like growth factor binding protein-1, insulin-like growth factor-I, and insulin-like growth factor-II in 16 patients with severe preeclampsia and 29 controls at the same gestational age were determined by an immunoradiometric assay and correlated with clinical parameters. Data were analyzed by t test and Pearson's method.

RESULTS

Insulin-like growth factor binding protein-1 was highly expressed on syncytiotrophoblasts, cytotrophoblasts, and decidual cells but not on placental fibroblasts. Immunostaining was greater at the maternal-fetal interface in severe preeclamptic patients compared with controls. Circulating insulin-like growth factor binding protein-1 levels in subjects with severe preeclampsia were 428.3 +/- 85.9 ng/ml compared with 76.6 +/- 11.8 in controls (p = 0.0007). Circulating insulin-like growth factor-I levels were 80.9 +/- 17.2 ng/ml compared with 179.4 +/- 28.2 ng/ml in controls (p = 0.0001). In contrast, insulin-like growth factor-II levels were not significantly different in the two groups. In subjects with severe preeclampsia insulin-like growth factor binding protein-1 levels correlated with diastolic blood pressure (r = 0.498, p 0.049) and aspartate transcarbamylase (0.621, p = 0.010).

CONCLUSIONS

The abundance of insulin-like growth factor binding protein-1 at the maternal-fetal interface in severely preeclamptic pregnancies suggests that the binding protein may participate in the pathogenesis of the shallow placental invasion observed in this disorder. Low circulating insulin-like growth factor-I and elevated insulin-like growth factor binding protein-1 levels may contribute to restricted placental and therefore fetal growth.

There are several <em>factors</em> like angiogenesis, lymphangiogenesis, genetic alterations, mutational <em>factors</em> that are involved in malignant transformation of potentially malignant oral lesions (PMOLs) to oral squamous cell carcinoma (OSCC). <em>Fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) is one of the prototypes of the large family of <em>growth</em> <em>factors</em> that bind heparin. FGF-2 induces angiogenesis and its receptors may play a role in synthesis of collagen. FGFs are involved in transmission of signals between the epithelium and connective tissue, and influence <em>growth</em> and differentiation of a wide variety of tissue including epithelia. The present study was undertaken to analyze expression of FGF-2 and its receptors FGFR-2 and FGFR-3 in 72 PMOLs, 108 OSCC and 52 healthy controls, and their role in risk assessment for malignant transformation of Leukoplakia (LKP) and Oral submucous fibrosis (OSMF) to OSCC. Immunohistochemistry was performed using antibodies against FGF-2, FGFR-2 and FGFR-3. IHC results were validated by Real Time PCR. Expression of FGF-2, FGFR-2 and FGFR-3 was upregulated from PMOLs to OSCC. While 90% (9/10) of PMOLs which showed malignant transformation (transformed) expressed FGF-2, only 24.19% cases (15/62) of PMOLs which were not transformed (untransformed) to OSCC expressed FGF-2. Similarly, FGFR-2 expression was seen in <em>16</em>/62 (25.81%) of untransformed PMOLs and 8/10 (80%) cases of transformed PMOLs. FGFR-3 expression was observed in 23/62 (37.10%) cases of untransformed PMOLs and 6/10 (60%) cases of transformed PMOLs. A significant association of FGF-2 and FGFR-2 expression with malignant transformation from PMOLs to OSCC was observed both at phenotypic and molecular level. The results suggest that FGF-2 and FGFR-2 may be useful as biomarkers of malignant transformation in patients with OSMF and LKP.

OBJECTIVE

Fibroblast growth factor 9 (FGF-9) is a relatively new member of the FGF family isolated from the conditioned medium of a human glioblastoma cell line as a secreting type factor that exhibits a growth-stimulating effect on primary glial cells. To elucidate the roles of FGF-9 in human brain tumors, the expression and biological activities of FGF-9 were studied using culture cells and surgically obtained tumor specimens.

METHODS

Measurement of FGF-9 and basic FGF in conditioned media of cell cultures was performed by using a sandwich enzyme immunoassay. The mitogenic effect of FGF-9 was evaluated by cell growth studies. FGF-9 expression in vivo was demonstrated by immunohistochemistry.

RESULTS

One of 4 glioma cell lines and 4 of 16 human meningiomas examined actually secreted detectable amounts of FGF-9 proteins. In comparison, basic FGF production was detected from 3 of 4 glioma cell lines and 11 of 16 human meningiomas. Similarly to basic FGF, recombinant human FGF-9 significantly stimulated the in vitro cell proliferation in three of four glioma cell lines investigated in a dose-dependent manner. A time course growth study using U87 MG cells revealed an accelerated growth stimulation by FGF-9 after Day 4. The growth stimulatory activity was also shown in three of four human meningiomas studied. Moderate to strong immunoreactivity for FGF-9 was observed in 40 (82%) of 49 human brain tumors examined irrespective of origin, tumor type, grade of malignancy, or whether initial or recurrent. In contrast, strong immunostaining was localized in neurons in the normal human cerebral cortex.

CONCLUSIONS

The present findings suggest that FGF-9 may be involved in the biology of human brain tumors with a possible importance in tumor cell growth. Whether the growth factor is more generally involved in oncogenesis of human tumors awaits further investigation.

BACKGROUND

Infantile hemangioma (IH) is the most common benign tumor in children with prevalence in the face and neck. Various treatment options including oral propranolol have been described for IH, but the mechanism of drugs remains enigmatic. The aim of this study was to investigate the pathogenesis and establish a reliable in vivo model of IH which can provide platform for drug exploration.

METHODS

Stem cells from the proliferating hemangiomas (HemSCs) were isolated by CD133-tagged immunomagnetic beads. Their phenotype and angiogenic property were investigated by flow cytometry, culturing on Matrigel, real-time polymerase chain reaction (PCR), immunofluorescent staining and injection into BALB/c-nu mice.

RESULTS

HemSCs had robust ability of proliferating and cloning. The time of cells doubling in proliferative phase was <em>16</em> hours. Flow cytometry showed that HemSCs expressed mesenchymal markers CD29, CD44, but not endothelial/hematopoietic marker of CD34 and hematopoietic marker CD45. The expression of CD105 was much lower than that of the reported hemangioma derived or normal mesenchymal stem cell (MSC). Real-time PCR showed that the mRNA levels of vascular endothelial <em>growth</em> <em>factor</em> (VEGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and matrix metalloproteinase-1 (MMP-1) of HemSCs were higher than that of neonatal human dermal <em>fibroblasts</em> (NHDFs) and human umbilical vein endothelial cells (HUVECs). After HemSCs were cultured on Matrigel in vitro, they formed tube-like structure in a short time (<em>16</em> hours) and differentiated into endothelial cells in 7 days. After 1 - 2 weeks of implantation into immunodeficient mice, HemSCs generated glucose transporter 1 positive blood vessels. When co-injected with HUVECs, the vascularization of HemSCs was greatly enhanced. However, the single implantation of HUVECs hardly formed blood vessels in BALB/c-nu mice (P < 0.05).

CONCLUSIONS

HemSCs may be some kinds of primitive mesoderm derived stem cells with powerful angiogenic ability, which can recapitulate human hemangioma by co-injecting into immunodeficient mice with HUVECs.

Phosphaturic mesenchymal tumors of the mixed connective tissue type (PMT) are very rare tumors of bone and soft tissues. Most patients with PMT have long-standing osteomalacia secondary to production of <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23), a hormone that inhibits phosphate reuptake within the renal proximal tubule. Previously, we have reported the detection of FGF23 mRNA in PMT by reverse transcription polymerase chain reaction (PCR); however, the low specificity and risk for nontumoral tissue contamination inherent in PCR-based methodology limit its clinical utility. We evaluated RNAscope as a semiquantitative method of in situ FGF23 mRNA detection in the diagnosis of PMT. Twenty-five PMTs (median 52 y, range 5 to 73 y) occurred in patients with tumor-induced osteomalacia (TIO), manifesting as masses (mean 3.9 cm, range 1.4 to 12 cm) in various bones and soft tissues. FGF23 mRNA was positive in 96% (22/23) informative cases of PMT: <em>16</em> cases scored 3+; 5 scored as 2+; 1 scored as 1+. Among these cases, FGF23 mRNA was detected in 3 malignant PMTs along with their metastases. Forty control cases included aneurysmal bone cyst (N=4), chondromyxoid fibroma (N=8), high-grade osteosarcomas (N=8), and (nonfamilial) tumoral calcinosis, as well as miscellaneous cartilage-forming tumors or osteoid-forming tumors and soft tissue tumors. All control cases were negative for FGF23 mRNA in the lesional cells. One aneurysmal bone cyst had rare FGF23 mRNA-expressing osteocytes clustered around remodeled bone. One ovarian serous carcinoma in a patient with disseminated disease, elevated serum FGF23, and TIO was negative for FGF23 mRNA in the neoplastic cells. We conclude that RNAscope is a highly sensitive and specific, semiquantitative in situ hybridization method of FGF23 mRNA detection applicable to formalin-fixed, paraffin-embedded tissues. Detection of FGF23 expression is a valuable diagnostic adjunct, especially in patients with occult TIO. Compared with reverse transcription PCR, this method preserves tissue morphology and reduces "false positives" related to detection of endogenous FGF23 mRNA expression by osteocytes.

BACKGROUND

The healing period of bone-implant osseointegration usually varies from 3 to 6 months or even longer. Failure may occur during this time. This study aimed to investigate whether osseointegration of dental implants can be enhanced by the combination of growth factors.

METHODS

Sixty-four implants were coated with polylactic acid and divided into four groups. Group I was applied with 1.0 mg recombinant human bone morphogenetic protein-2 (rhBMP-2) and 200 microg recombinant human basic fibroblast growth factor (rhbFGF), group II with 1.0 mg rhBMP-2 and 250 mug recombinant human insulin-like growth factor-I (rhIGF-I), group III with 1.0 mg rhBMP-2, and group IV without growth factors as control. In total, 16 rabbits were used, and two osteotomies were drilled on each side of the femur, in which four different groups were randomly placed. Four weeks after implanting, 20 mg calcein green/kg body weight was administered intravenously, and 8 weeks after implanting, 20 mg alizarin/kg body weight was administered intravenously. Twelve weeks after implanting, the animals were sacrificed. The block of bone with implants was embedded in methylmethacrylate and sectioned, and the percentage of new bone surrounding the implant was analyzed by confocal laser scanning microscopy.

RESULTS

There was a statistical difference in bone formation between rhBMP-2-applied groups and the non-applied group at 4 or 8 weeks, and no significant difference between groups I and II (although bone formation in group II was greater than that in group I at 4 weeks). The bone formation in group II was greater than that in group III at 4 or 8 weeks. The formed bone in group I was also greater than the one in group III at 8 weeks, but there was no difference at 4 weeks.

CONCLUSIONS

rhBMP-2 could increase new bone formation, and it acted synergistically with rhbFGF and rhIGF-I to improve bone-implant osseointegration. The combination of rhBMP-2 and rhbFGF (group 1) showed faster growth of new bone than other groups at 8 months.

BACKGROUND

Few studies have yet addressed the effects of di(2-ethylhexyl) phthalate (DEHP) in house dust on human nasal mucosa.

OBJECTIVE

We investigated the effects of house dust containing DEHP on nasal mucosa of healthy and house dust mite (HDM)-allergic subjects in a short-term exposure setting.

METHODS

We challenged <em>16</em> healthy and <em>16</em> HDM-allergic subjects for 3 hr with house dust at a concentration of 300 microg/m(3) containing either low (0.41 mg/g) or high (2.09 mg/g) levels of DEHP. Exposure to filtered air served as control. After exposure, we measured proteins and performed a DNA microarray analysis.

RESULTS

Nasal exposure to house dust with low or high DEHP had no effect on symptom scores. Healthy subjects had almost no response to inhaled dust, but HDM-allergic subjects showed varied responses: DEHP(low) house dust increased eosinophil cationic protein, granulocyte-colony-stimulating factor (G-CSF), interleukin (IL)-5, and IL-6, whereas DEHP(high) house dust decreased G-CSF and IL-6. Furthermore, in healthy subjects, DEHP concentration resulted in 10 differentially expressed genes, whereas <em>16</em> genes were differentially expressed in HDM-allergic subjects, among them anti-Müllerian hormone, which was significantly up-regulated after exposure to DEHP(high) house dust compared with exposure to DEHP(low) house dust, and fibroblast growth factor 9, IL-6, and transforming growth factor-beta1, which were down-regulated.

CONCLUSIONS

Short-term exposure to house dust with high concentrations of DEHP has attenuating effects on human nasal immune response in HDM-allergic subjects, concerning both gene expression and cytokines.

Thrombin is a powerful agonist for a variety of cellular responses including platelet aggregation and vascular smooth muscle cell (SMC) proliferation. These actions are mediated by a thrombin receptor known as protease-activated receptor-1 (PAR-1). Recently we discovered that 1-(3-tert-butyl-4-methoxy-5-morpholinophenyl)-2-(5,6-diethoxy-7-fluoro-1-imino-1,3-dihydro-2H-isoindol-2-yl)ethanone hydrobromide (E5555, atopaxar) is a potent and selective thrombin receptor antagonist. This study characterized the pharmacological effects of E5555 on SMC proliferation in vitro and in a rat model of intimal thickening after balloon injury in vivo. E5555 selectively inhibited rat aortic SMC proliferation induced by thrombin and thrombin receptor-activating peptide (TRAP) with half maximal inhibitory concentration (IC(50)) values of 0.<em>16</em> and 0.038 μM, respectively. E5555 did not inhibit rat SMC proliferation induced by basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and platelet-derived <em>growth</em> <em>factor</em> (PDGF) at concentrations up to 1μM. In addition, E5555 inhibited human aortic SMC proliferation induced by thrombin at concentrations of 0.3 and 3units/ml with IC(50) values of 0.028 and 0.079 μM, respectively, whereas it did not affect bFGF-induced proliferation at concentrations up to 1μM. Repeated oral administration of 30 mg/kg E5555 (once daily for <em>16</em> days) significantly reduced neointimal formation in the balloon-injured rat arterial model. These results suggested that a PAR-1 antagonist could be effective for treating restenosis following vascular intervention in addition to preventing thrombus formation. E5555 could thus have therapeutic potential for restenosis and chronic atherothrombotic disease.

A composite procedure involving molecular modelling and a property-pattern algorithm, the Resonant Recognition Model (RRM), has been applied to structure-function studies with basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). Property-pattern characteristics for biological activity and receptor recognition for a group of FGF-related proteins were defined and then used to aid the design of a set of peptides which can act as bFGF antagonists. Molecular modelling techniques were then employed to identify the peptide within this set with the greatest conformational similarity to the putative receptor domain of bFGF. This <em>16</em> amino acid residue peptide (<em>16</em>mer), which exhibits no sequence homology to bFGF, antagonised the stimulatory effect of bFGF on <em>fibroblast</em> [3H]thymidine incorporation and cell proliferation, but exerted no effect itself in these in vitro bioassays.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) is important in development, wound healing and angiogenesis. The human plasma proteinase inhibitor alpha2-macroglobulin (alpha2M) binds to and regulates the biological activity of various <em>growth</em> <em>factors</em>, including FGF-2. FGF-2 binds specifically and saturably to native alpha2M and conformationally modified alpha2M (alpha2M*); however, the KD for FGF-2 binding to alpha2M* is 10-fold lower. This study investigates the biochemical nature of the interaction between FGF-2 and alpha2M* and localizes a possible FGF-2 binding site in the alpha2M subunit. FGF-2 binding to alpha2M* was not affected by shifts in pH between 6.5 and 10; however, increasing temperature decreased the KD for this interaction. The binding affinity of FGF-2 for alpha2M* also increased with increasing ionic strength. These results are consistent with the hypothesis that hydrophobic interactions predominate in promoting FGF-2 association with alpha2M*. Consistent with this hypothesis, FGF-2 bound to a glutathione S-transferase fusion protein containing amino acids 591-774 of the alpha2M subunit (FP3) and to a hydrophobic <em>16</em>-amino-acid peptide (amino acids 718-733) within FP3. Specific binding of FGF-2 to the <em>16</em>-amino-acid peptide was inhibited by excess transforming <em>growth</em> <em>factor</em>-beta1. When the <em>16</em>-amino-acid peptide was chemically modified to neutralize the only two charged amino acids, FGF-2-binding activity was unaffected, supporting the predominant role of hydrophobic interactions. FGF-2 presentation to signalling receptors is influenced by <em>growth</em> <em>factor</em> binding to heparan sulphate proteoglycans (HSPGs), which is electrostatic in nature. Our results demonstrate that the interactions of FGF-2 with alpha2M* and HSPGs are biochemically distinct, suggesting that different FGF-2 sequences are involved.

Combinational pharmacotherapy with individually efficacious agents is a potential strategy for the treatment of traumatic central nervous system (CNS) injury. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) has been shown to be neuroprotective against excitotoxic, ischemic, and traumatic injury to the CNS, while acute posttraumatic treatment with magnesium (Mg2+) has been shown to decrease the motor and cognitive deficits following experimental brain injury. In this study, bFGF and Mg2+ were evaluated separately and in combination to assess their potential additive effects on posttraumatic neurological recovery and histological cell loss (lesion volume). Twenty minutes after fluid percussion (FP) brain injury of moderate severity (2.2-2.4 atm), anesthetized rats received a 15-min intravenous infusion of either 125 mumol of MgCl2 or vehicle, followed 5 min later by a 24-h constant intravenous infusion of either bFGF (<em>16</em> micrograms/h) or vehicle. Injured animals had a significant motor deficit when compared to sham (uninjured) animals at both 48 h and 7 days postinjury. At 48 h postinjury, there were no significant differences among injured animals when compared by treatment. By 7 days postinjury, injured animals treated with MgCl2 alone displayed significantly improved motor function when compared to brain-injured, vehicle-treated animals (p < 0.05). Animals treated with either bFGF alone or a combination of MgCl2 and bFGF displayed no significant neurological improvement relative to vehicle-treated injured animals at 7 days. No effect of any drug treatment of combination was observed on the extent of the postinjury lesion volume in the injured cortex. These results suggest that caution must be exercised when combining "cocktails" of potentially neuroprotective compounds in the setting of traumatic brain injury.