BACKGROUND

Von Hippel Lindau disease (VHL) is a rare autosomal dominant inherited disorder characterized by highly vascularized tumors in various organs. The abundant presence of endothelial cells in VHL tumors strongly suggest a role of the VHL tumor suppressor gene in the regulation of angiogenesis. Recently, in vitro studies have shown that the VHL tumor suppressor gene regulates the expression of vascular endothelial growth factor (VEGF). We investigated whether VHL patiens have increased levels of VEGF in their body fluids.

METHODS

The concentration of VEGF was measured in fluid of the anterior chamber of the eye, serum, urine, and fluid from renal cysts of VHL patients and unaffected individuals by ELISA. In addition, levels of basic fibroblast growth factor (bFGF), interleukin-8 (IL-8) and endothelin-1 (ET-1) were measured in urine and serum of VHL patients and control subjects.

RESULTS

In 80% of the VHL patients VEGF was detectable in aqueous fluid of the anterior chamber of their eyes. A strong positive correlation (r = 0.90) was found between the age of VHL patients and ocular VEGF concentrations. At comparable age, VEGF levels in ocular fluid of VHL patients were significantly higher (P < 0.001) than in unaffected subjects. No correlation was found between VEGF concentration and the presence of retinal angiomas. A 10 and 16 fold increase of VEGF concentration was seen in fluid from two independent VHL-related cysts as compared with VEGF serum levels of the same patient. The mean concentration of VEGF in serum of VHL patients (n = 15) (319 +/- 84 pg/ml) was higher than in matched controls (238 +/- 68 pg/ml; P = NS). The mean concentration of VEGF in urine of VHL patients (128 +/- 36 pg/ml) was lower than in matched controls (183 +/- 25 pg/ml; P = NS). Concentrations of VEGF did not correlate with the presence of VHL-related tumors. No differences were observed between concentrations of bFGF, IL-8 and ET-1 in serum and urine of VHL patients and matched controls.

CONCLUSIONS

These findings support a role for the VHL tumor suppressor gene in the in vivo regulation of VEGF.

We have purified acidic and basic <em>fibroblast</em> <em>growth</em> <em>factors</em> (c-aFGF, c-bFGF) from 11 day-old chick embryo brain, retina and vitreous by heparin-Sepharose chromatography and reverse phase HPLC. The analysis of their biological activity as well as their molecular weight indicates that they were analogous to basic or acidic human and bovine FGF. The ratio of c-aFGF to c-bFGF activity depended of the tissue. In brain c-aFGF represented 66% of the total mitogenic activity retained on the heparin-sepharose column and c-bFGF 34% while retina contained <em>16</em>% of c-aFGF and 84% of c-bFGF; vitreous 78% of c-aFGF and 22% of c-bFGF. Like human aFGF, Heparin stimulated purified c-aFGF mitogenic activity in the absence of serum but inhibited the activity of the retina acid soluble extract, in the presence of foetal calf serum (FCS). Thus, chick embryo and adult human acidic and basic FGF respectively share the same biochemical properties. Since there are no blood vessels in chick retina or vitreous, their presence in these tissues suggests that angiogenesis is not the only role of these <em>growth</em> <em>factors</em>.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) has been purified 333,000-fold from human placenta by a combination of salt precipitation, cation-exchange chromatography, and Heparin-Sepharose affinity chromatography. Molecular weight (15-<em>16</em> kDaltons), amino acid composition, bioactivity and immunological crossreactivity with bovine pituitary FGF indicate that the mitogens from the two species are closely related molecules.

The E5 protein of bovine papillomavirus is a 44-amino acid, Golgi-resident, type II transmembrane protein that efficiently transforms immortalized mouse <em>fibroblasts</em>. The transmembrane (TM) domain of E5 is not only critical for biological activity, it also regulates interactions with cellular targets including the platelet derived <em>growth</em> <em>factor</em> receptor (PDGF-R) and the <em>16</em>-kDa subunit of the vacuolar proton ATPase (V-ATPase). In order to define the specific TM amino acids essential for E5 biological and biochemical activity, we performed scanning alanine mutagenesis on 25 of the 30 potential TM residues and genetically mapped discrete alpha-helical domains which separately regulated the ability of E5 to bind PDGF-R, activate PDGF-R, and to form oligomers. Alanine substitutions at positions 17, 21, and 24 (which lie on the same helical face) greatly inhibited E5 association with the PDGF-R, suggesting that this region comprises the receptor binding site. PDGF-R activation also mapped to a specific but broader domain in E5; mutant proteins with alanines on one helical face (positions 8, 9, 11, <em>16</em>, 19, 22, and 23) continued to induce PDGF-R tyrosine phosphorylation, whereas mutant proteins with alanines on the opposite helical face (positions 7, 10, 13, 17, 18, 21, 24, and 25) did not, indicating that the latter helical face was critical for mediating receptor transphosphorylation. Interestingly, these "activation-defective" mutants segregated into two classes: 1) those that were unable to form dimers but that could still form higher order oligomers and transform cells, and 2) those that were defective for PDGF-R binding and were transformation-incompetent. These findings suggest that the ability of E5 to dimerize and to bind PDGF-R is important for receptor activation. However, since several transformation-competent E5 mutants were defective for binding and/or activating PDGF-R, it is apparent that E5 must have additional activities to mediate cell transformation. Finally, alanine substitutions also defined two separate helical faces critical for E5/E5 interactions (homodimer formation). Thus, our data identify distinct E5 helical faces that regulate homologous and heterologous intramembrane interactions and define two new classes of biologically active TM mutants.

Polymerase chain reaction analysis revealed an mRNA in rat prostate that results from alternate splicing of exon <em>16</em> in the heparin-binding <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor kinase type 2 gene (FGFR2). The absence of exon <em>16</em> and the shift in reading frame at the exon 15-17 junction predicts an expression product (FGFR2-2) with a unique COOH-terminus that does not exhibit the major autophosphorylation site (tyrosine 789) required for interaction of phospholipase C gamma 1 with the full-length FGFR2-1 isoform. Nuclease protection analysis revealed that the FGFR-2 splice variant is expressed in quantities equal to or greater than the FGFR2-1 isoform in normal prostate tissue. When combined with the same FGFR2 extracellular domain, co-expression of the two COOH-terminal variants may mediate effect of the same FGF ligand on different signal transduction pathways.

The effects of transforming <em>growth</em> <em>factor</em> beta 1 (TGF beta 1) on DNA synthesis and functional differentiation of astroglial cells cultured in serum-free medium were investigated. TGF beta 1 diminished and delayed the peak of DNA synthesis induced by serum. TGF beta 1-treated cells were larger than control cells. This <em>factor</em> delayed the appearance of process-bearing cells induced by acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> treatment and also affected the astrocyte-specific enzyme glutamine synthetase (GS), whose accumulation is under hydrocortisone (HC) control. TGF beta 1 inhibited the induction of GS activity by HC in a dose- and time-dependent manner. Moreover, pretreatment with TGF beta 1 for 4 h maintained the inhibition of GS activity for approximately <em>16</em> h after removal of this <em>factor</em> from culture medium. These results suggest that TGF beta 1 may be an important regulator of astrocyte <em>growth</em> and differentiation.

We reported previously that a protein tyrosine phosphatase (PTP) activity is associated with the immunoprecipitated hepatocyte <em>growth</em> <em>factor</em> (HGF) receptor, also known as Met. The activity increased reversibly when Met was stimulated by HGF and decreased when Met was inactivated by PMA. To identify the PTP-binding region, we used deletion mutants of the receptor beta-subunit. The PTP activity did not associate with Tpr-Met, a construct containing residues 1010-1390 of Met fused to Tpr. In contrast, PTP activity was present when the expressed protein contained the full juxtamembrane region (residues 956-1390 of Met) or part of this region (residues 957-1390 or 995-1390), indicating that the PTP-binding region is between residues 995 and 1009. This region includes Tyr1003, a site involved in Met downstream signalling. Incubation of Met immunoprecipitated from GTL-<em>16</em> cells with an 8-mer phosphopeptide derived from residues 1003-1010 induced a marked decrease in the associated PTP activity, suggesting that the peptide reproduced the PTP-binding region. Mutation of Glu, Asp or Arg at positions -4, -1 or +1 respectively relative to Tyr1003 in a 9-mer peptide (residues 999-1007) abolished the ability of the peptide to decrease the PTP activity associated with Met. Phosphorylation of Tyr1003 was not required for PTP binding, since: (1) both phospho- and dephospho-peptides on a solid bead bound PTP activity from a GTL-<em>16</em> cell extract, and (2) PTP activity was associated with a Met deletion mutant lacking residues 1-955 in which Tyr1003 had been changed into Phe. In order to partially purify the PTP from the GTL-<em>16</em> cell extract, an affinity column was prepared using the Met-derived peptide comprising residues 998-1007. Less than 0.1% of the total cellular PTP was retained by the column, and was eluted with low salt concentrations. Using antibodies, this PTP was identified as PTP-S, a soluble PTP present in epithelial cells and <em>fibroblasts</em>.

Liver fibrosis is a complex process characterized by two major events: fibroproliferation and increased collagen synthesis. The exact role of cytokines in the pathogenesis of hepatic fibrosis remains to be established, but platelet-derived <em>growth</em> <em>factor</em> clearly stimulates proliferation of <em>fibroblasts</em> and increases collagen synthesis. In in vitro studies, pentoxifylline, a methylxanthine, significantly reduced platelet-derived <em>growth</em> <em>factor</em>-driven proliferation of <em>fibroblasts</em>. Platelet-derived <em>growth</em> <em>factor</em> has also been identified as a fibroproliferative <em>factor</em> produced spontaneously by monocytes obtained from patients with liver disease. Long-term administration of pentoxifylline (<em>16</em> mg/kg orally, 5 days/wk for 12 wk) in an animal model of liver fibrosis prevented elevations in gamma-glutamyl transpeptidase and alkaline phosphatase levels and prevented the reduction in serum albumin level normally observed in this animal model of liver disease. The animal model used was a long-term, low-dose yellow phosphorus--induced model in pigs that reproducibly results in extensive fibrosis after 10 to 12 wk of treatment. Long-term administration of pentoxifylline also prevented the histological changes characteristic of fibrosis in this animal model. Collagen concentration was significantly elevated in liver sections obtained from animals receiving yellow phosphorus, compared with controls. Long-term pentoxifylline treatment resulted in significantly lower collagen concentrations in liver sections from animals receiving yellow phosphorus than in sections from animals receiving yellow phosphorus alone; this was supported by histological observation. Therefore administration of pentoxifylline prevented the biochemical and histological changes associated with an animal model of liver disease. Pentoxifylline will likely have an important therapeutic role in liver fibrosis.

OBJECTIVE

To investigate the role of urinary measurements of an angiogenic factor, basic fibroblast growth factor (bFGF), in the assessment of patients with bladder cancer.

METHODS

Urine from 83 patients was assayed using a commercially available ELISA for bFGF. Thirty-eight patients had a bladder tumour and 21 had a history of bladder cancer but no disease at the time of testing. Twenty-four patients acted as controls, 16 of whom were about to undergo transurethral resection of the prostate (TURP) for benign prostatic hypertrophy (BPH) and eight who had no urological disease.

RESULTS

Median urinary bFGF was higher in patients with active bladder cancer than in those with a clear cystoscopy (5.20 and 2.13 ng/g creatinine, respectively; P < 0.005). Median urinary bFGF was also elevated in patients about to undergo TURP (4.52 ng/g creatinine). Using a threshold value of 6.0 ng/g creatinine, the sensitivity of the test for detecting cancer was 42% and specificity was 88%. At a threshold value of 4.0 ng/g the sensitivity was 62% and the specificity 70%.

CONCLUSIONS

The relationship between urinary basic FGF and the presence of bladder cancer was significant. The test is not sufficiently sensitive or specific to use as a screening test for bladder cancer but may be very useful in monitoring the effectiveness of systemic therapies in bladder cancer. Elevated levels of bFGF in the urine of patients about to undergo TURP suggests a role for bFGF in the pathogenesis of BPH.

OBJECTIVE

Fibroblast activity promotes adverse left ventricular (LV) remodeling that underlies the development of ischemic cardiomyopathy. Transforming growth factor-β (TGF-β) is a potent stimulus for fibrosis, and the extracellular signal-regulated kinases(ERK) 1/2 pathway also contributes to the fibrotic response. The thrombin receptor, protease-activated receptor 1 (PAR1), has been shown to play an important role in the excessive fibrosis in different tissues. The aim of this study was to investigate the influence of a PAR1 inhibitor, SCH79797, on cardiac fibrosis, tissue stiffness and postinfarction remodeling, and effects of PAR1 inhibition on thrombin-induced TGF-β and (ERK) 1/2 activities in cardiac fibroblasts.

METHODS

We used a rat model of myocardial ischemia-reperfusion injury, isolated cardiac fibroblasts, and 3-dimensional (3D) cardiac tissue models fabricated to ascertain the contribution of PAR1 activation on cardiac fibrosis and LV remodeling.

RESULTS

The PAR1 inhibitor attenuated LV dilation and improved LV systolic function of the reperfused myocardium at 28 days. This improvement was associated with a nonsignificant decrease in scar size (%LV) from 23 ± % in the control group (n = 10) to 16% ± 5.5% in the treated group (n = 9; P = .052). In the short term, the PAR1 inhibitor did not rescue infarct size or LV systolic function after 3 days. The PAR1 inhibition abolished thrombin-mediated ERK1/2 phosphorylation, TGF-β and type I procollagen production, matrix metalloproteinase-2/9 activation, myofibroblasts transformation in vitro, and abrogated the remodeling of 3D tissues induced by chronic thrombin treatment.

CONCLUSIONS

These studies suggest PAR1 inhibition initiated after ischemic injury attenuates adverse LV remodeling through late-stage antifibrotic events.

The antiangiogenic activity of the multidomain plasma protein histidine-proline-rich glycoprotein (HPRG) is localized to its histidine-proline-rich (H/P) domain and has recently been shown to be mediated, at least partially, through binding to cell-surface tropomyosin in <em>fibroblast</em> <em>growth</em> <em>factor</em>-2-activated endothelial cells (X. Guan et al., Thromb Haemost, in press). HPRG and its H/P domain, but not the other domains of HPRG, bind specifically and with high affinity to tropomyosin. In this study, we characterize the interaction of the H/P domain with tropomyosin and delineate the region within the H/P domain responsible for that interaction. The H/P domain of HPRG consists mostly of repetitions of the consensus sequence [H/P][H/P]PHG. Applying an in vitro tropomyosin binding assay, we demonstrate that the synthetic peptide HHPHG binds to tropomyosin in vitro and inhibits angiogenesis and tumor <em>growth</em> in vivo. The affinity for tropomyosin increases exponentially upon multimerization of the HHPHG sequence, with a concurrent increase in antiangiogenic activity. Specifically, the tetramer (HHPHG)4 has significant antiangiogenic activity in the Matrigel plug model (IC50 approximately 600 nm) and antitumor effects in two syngeneic mouse tumor models. Thus, we show that a <em>16</em>-mer peptide analogue mimics the antiangiogenic activity of intact HPRG and is also able to inhibit tumor <em>growth</em>, suggesting that cell surface tropomyosin may represent a novel antiangiogenic target for the treatment of cancer.

We reported previously that an N-terminally truncated insulinlike <em>growth</em> <em>factor</em> I receptor (IGFR) fused to avian sarcoma virus UR2 gag p19 had a greater transforming potential than did the native IGFR, but it failed to cause tumors in vivo. To investigate whether the 36 amino acids (aa) of the IGFR extracellular (EC) sequence in the gag-IGFR fusion protein encoded by the retrovirus UIGFR have a modulatory effect on the biological and biochemical properties of the protein, four mutants, NM1, NM2, NM3, and NM4 of the EC sequence were constructed. NM1 lacks the entire 36 aa residues; NM2 lacks the N-terminal <em>16</em> aa residues (aa 870 to 885), including two potential N-linked glycosylation sites of the EC sequence; NM3 contains a deletion of the C-terminal 20 aa residues (aa 886 to 905) of the EC sequence; and NM4 contains N-to-Q substitutions at both N-linked glycosylation sites. NM1 was the strongest of the four mutants in promoting anchorage-independent <em>growth</em> of transfected chicken embryo <em>fibroblasts</em>, while NM2 and NM4 had weaker transforming potential than did the original UIGFR virus. Only NM1 and NM3 were able to induce sarcomas in chickens. The four NM mutant-transformed cells expressed the expected proteins with comparable steady-state levels. The in vitro tyrosine kinase activity of P53NM1 was about fourfold higher than that of the parental P57-75UIGFR, whereas NM2 and NM4 proteins exhibited four- to fivefold-lower kinase activities. Despite lacking the IGFR EC sequence, P53NM1 formed covalent dimers similar to those formed by the parental P57-75UIGFR. Increased phosphatidylinositol (PI) 3-kinase activity was found to be associated with the mutant IGFR proteins. Among NM4 proteins. Elevated tyrosine phosphorylation of cellular proteins of 35, 120, 140, <em>16</em>0, and 170 kDa was detected in all mutant IGFR-transformed cells. We conclude that the EC 36-aa sequence of IGFR in the gag-IGFR fusion protein exerts intricate modulatory effects on the protein's transforming and tumorigenic potential. The 20 aa residues immediately upstream of the transmembrane domain have an inhibitory effect on the tumorigenic potential of gag-IGFR, whereas N-linked glycosylation within the EC sequence appears to have a positive effect on the transforming potential of UIGFR. Increased in vitro kinase activity and, to a lesser extent, in vivo tyrosine phosphorylation as well as the elevated association of PI 3-kinase activity with IGFR proteins seem to be correlated with the transforming potential of IGFR mutant proteins.

BACKGROUND

The association between oral squamous cell carcinoma (OSCC) and viral and chemical factors is uncertain. Therefore the correlation of viral and chemical factors with oral cancer in Taiwan was investigated.

METHODS

Thirty-seven paraffin-embedded oral cancer biopsies and 36 normal oral tissue specimens were examined by the polymerase chain reaction method for six viruses: HPV, CMV, EBV, HSV-1, HSV-2 and HHV-8. To elucidate the role of arecoline in the oncogenesis of oral cancer, human buccal fibroblasts, oral submucosal fibroblasts and three cancer cell lines KB, GNM and TSCCa were used for MTT cytotoxity assay and flow cytometry DNA content analysis.

RESULTS

Two (5.4%) HSV-1-positive and four (10.8%) HPV-positive cases were recognized in oral cancer biopsies. Among the four HPV-positive tissues, two were further typed as HPV-16, one was identified as HPV-18- and HSV-1-positive; and one contained both HPV-16 and HPV-18. One sample presented HSV-1 only. Arecoline, at a concentration lower than 0.8 micro g/ml, increased cell growth (all cell types); at higher concentrations (25-400 micro g/ml) it was cytotoxic. The cell cycle was demonstrated to be altered either by low or high concentrations of arecoline treatment, depending on the cells treated.

CONCLUSIONS

The data demonstrated that HPV, HSV-1 and betel quid chewing were significantly associated with OSCC, but HSV-2, CMV, EBV and HHV-8 were not. We suggest that the most determinative factor for oral cancer may be chemical in nature rather than viral infection.

Heparin was previously reported to potentiate the mitogenic activity of endothelial cell mitogens in a crude extract of bovine hypothalami (Thornton, S. C., Mueller, S. N., and Levine, E. M. (1983) Science 222, 623-625). We and others (Gospodarowicz, D., and Cheng, J. (1986) J. Cell. Physiol. 128, 475-484) have reported that the <em>growth</em> stimulatory effects of acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) are potentiated in a similar manner. We have used these observations as the basis of an assay to characterize the importance of size, sulfation, and anticoagulant activity of heparin in mediating this effect. Partial nitrous acid depolymerization of heparin from porcine intestinal mucosa resulted in a mixture of heparin fragments, containing oligosaccharides ranging from disaccharides to polysaccharides of about 40 monosaccharides in length. This mixture was fractionated by ion exchange chromatography and gel permeation chromatography to obtain size-homogeneous oligosaccharides with different degrees of sulfation. Assay of these heparin-derived saccharides in the presence of a suboptimal concentration of aFGF revealed that a minimum chain length and a certain degree of sulfation is required in order to potentiate the action of aFGF. Low sulfate oligosaccharides (4-<em>16</em> units) were unable to potentiate aFGF, whereas medium sulfate fractions of octadecasaccharides and larger were able to moderately potentiate aFGF. The potentiation of aFGF by the high sulfate fraction correlated with the saccharide size: 12 or more monosaccharide units were necessary to achieve potentiation equivalent to whole heparin, octa- and decasaccharides were mildly stimulatory, and hexasaccharides were without effect. In the absence of aFGF, intact heparin as well as all the oligosaccharides examined, inhibited the proliferation of capillary endothelial cells to approximately the same degree, between 20 and 50% inhibition. When a tetradecasaccharide was separated into a binding and a nonbinding fraction on matrix-bound antithrombin III, no difference was seen for these fractions in the endothelial cell proliferation assay. These results indicate that both size and sulfation of a heparin-derived oligosaccharide contribute to its ability to interact with aFGF and/or endothelial cells and that this interaction is independent of anticoagulant activity. In addition, our findings suggest that the inhibitory and potentiating effects of heparin on capillary endothelial cells have different structural requirements.

OBJECTIVE

Newborns with congenital diaphragmatic hernia (CDH) still have a high mortality rate, which has been attributed to pulmonary hypoplasia and pulmonary hypertension. Fibroblast growth factors (FGFs) are essential components of the gene network that regulates lung development. Recent studies suggest that the new member of FGF family, FGF-10, plays a fundamental role in branching morphogenesis and is essential for lung formation. FGF-10-deficient mice exhibit complete absence of lungs. FGF-7 promotes epithelial proliferation and expansion leading to the formation of cystlike structures. The aim of this study was to determine the gene level expression of FGF-10 and FGF-7 in the lung of nitrofen-induced CDH.

METHODS

Congenital diaphragmatic hernia (CDH) was induced in pregnant rats after administration of 100 mg of nitrofen on day 9.5 of gestation (term, 22 days). In control animals, the same dose of olive oil was given without nitrofen. Cesarean section was performed on day 21 of gestation. The fetuses were divided into 3 groups: normal controls (n = 16), nitrofen induced without CDH (n = 16), and nitrofen-induced CDH (n = 16). Total RNA and DNA were extracted from the lung in each group and measured. mRNA was extracted from total RNA. Reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate mRNA expressions of FGF-10 and FGF-7. Levels of mRNA were expressed as a ratio of the band density divided by that of beta-actin, a house-keeping gene.

RESULTS

FGF-10 mRNA expression was decreased significantly in CDH lung (2.914 +/- 0.320) compared with controls (4.062 +/- 0.307; P <.05) and nitrofen induced without CDH lung (3.923 +/- 0.250; P <.01). FGF-7 mRNA expression was decreased significantly in CDH lung (0.777 +/- 0.097) compared with controls (1.028 +/- 0.093; P <.01).

CONCLUSIONS

Decreased gene expression of FGF-10 and FGF-7 in the hypoplastic lung suggests that pulmonary hypoplasia in nitrofen-induced CDH rat may be caused by reduced synthesis of FGF-10 and FGF-7 during lung morphogenesis.

The effects of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) on the development of hair follicles in neonatal mouse skin were examined. Newborn mice (B6C3-based strain) were given daily subcutaneous injections of bFGF and bovine serum albumin (BSA) for 7 days. By Day 9, distinct areas of hairless, unpigmented skin were observed surrounding the sites treated with bFGF. This phenomenon persisted until about 14-<em>16</em> days of age when the emergence of hair in the bald patches was first seen. Hair <em>growth</em> continued in the treated regions until they were mostly covered at 18-20 days of postnatal age. Control sites injected with BSA only were indistinguishable from the surrounding pigmented, hairy skin at all ages. Histological examination of skin from various ages of bFGF-treated mice suggested that the <em>growth</em> <em>factor</em> had affected both the initiation and the development of the hair follicles. This resulted in a significant delay in the first and subsequent hair cycles when compared to control animals injected with BSA only.

Phosphaturic mesenchymal tumor, mixed connective tissue type (PMTMCT) is a rare neoplasm that can cause tumor-induced osteomalacia due to overproduction of a phosphaturic hormone, <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23). We report here a case of subcutaneous PMTMCT, non-phosphaturic variant, in the sole. We also review 32 Japanese cases of PMTMCT reported in detail. They occurred in <em>16</em> men and 15 women (one was unknown), with ages ranging 20-73 years (median, 48). Tumors were found in soft tissue, bone and sinuses in 17, 11 and four, respectively. A history of long-standing osteomalacia was noted in all cases except two non-phosphaturic variant cases. Serum FGF23 level was elevated in 11 of 12 cases examined. In terms of follow-up information, metastases were found in four patients, and two patients died of disease. In conclusion, PMTMCT is histologically a benign lesion; however, there may be rare metastatic and malignant cases. Wider recognition of the histological features of this unique neoplasm would aid its distinction from the large number of mesenchymal tumors for which it may be mistaken and should enable correct diagnosis of tumors with osteomalacia.

We report here a phase 1 study of LY2874455, a potent oral selective pan-fibroblast growth factor receptor (FGFR) inhibitor.

The primary objective was to determine the recommended phase 2 dosing (RP2D). Secondary objectives included determining toxicity, antitumor activity, pharmacokinetics (PK), and pharmacodynamic (PD) properties of LY2874455.

This study comprised two parts: (a) dose escalation with 3 + 3 cohorts in patients with solid tumors and (b) dose-expansion cohorts in patients with gastric cancer (GC) and non-small cell lung cancer (NSCLC). Part A: 36 patients in 11 dose cohorts ranging from 2 to 24 mg twice daily (BID). RP2D was 16 mg BID. Part B: GC cohort, 29 patients, NSCLC cohort, 27 patients, all treated at the RP2D.

LY2874455 was slowly absorbed and generally showed linear PK. The effective half-life was ∼12 h. PD properties of LY2874455 occurred at doses ≥10 mg by increases in serum phosphorus. Phosphate binders were administered to control serum phosphorus. LY2874455 was generally well tolerated; most toxicities were grade 1 or 2; most frequent were hyperphosphatemia, diarrhea, and stomatitis.

part A: 24 patients evaluable: 1 patient in the 14-mg BID cohort with GC had a partial response (PR); 14 patients had stable disease (SD); part B: NSCLC cohort: 11 of 12 evaluable patients had SD; GC cohort: 15 patients evaluable: 1 patient with PR; 12 patients with SD.

LY2874455 has an RP2D of 16 mg BID and demonstrated good tolerability and activity in solid-organ cancer patients. The role of FGFR inhibition on tumor growth in patients requires further study. (NCT01212107).

In April 2008, the Medical Advisory Secretariat began an evidence-based review of the literature concerning pressure ulcers.Please visit the Medical Advisory Secretariat Web site, http://www.health.gov.on.ca/english/providers/program/mas/tech/tech_mn.html to review these titles that are currently available within the Pressure Ulcers series.PRESSURE ULCER PREVENTION: an evidence based analysisThe cost-effectiveness of prevention strategies for pressure ulcers in long-term care homes in Ontario: projections of the Ontario Pressure Ulcer Model (field evaluation)MANAGEMENT OF CHRONIC PRESSURE ULCERS: an evidence-based analysis

OBJECTIVE

The Medical Advisory Secretariat (MAS) conducted a systematic review on interventions used to treat pressure ulcers in order to answer the following questions: Do currently available interventions for the treatment of pressure ulcers increase the healing rate of pressure ulcers compared with standard care, a placebo, or other similar interventions?Within each category of intervention, which one is most effective in promoting the healing of existing pressure ulcers?

BACKGROUND

A pressure ulcer is a localized injury to the skin and/or underlying tissue usually over a bony prominence, as a result of pressure, or pressure in conjunction with shear and/or friction. Many areas of the body, especially the sacrum and the heel, are prone to the development of pressure ulcers. People with impaired mobility (e.g., stroke or spinal cord injury patients) are most vulnerable to pressure ulcers. Other factors that predispose people to pressure ulcer formation are poor nutrition, poor sensation, urinary and fecal incontinence, and poor overall physical and mental health. The prevalence of pressure ulcers in Ontario has been estimated to range from a median of 22.1% in community settings to a median of 29.9% in nonacute care facilities. Pressure ulcers have been shown to increase the risk of mortality among geriatric patients by as much as 400%, to increase the frequency and duration of hospitalization, and to decrease the quality of life of affected patients. The cost of treating pressure ulcers has been estimated at approximately $9,000 (Cdn) per patient per month in the community setting. Considering the high prevalence of pressure ulcers in the Ontario health care system, the total cost of treating pressure ulcers is substantial.

METHODS

Wounds normally heal in 3 phases (inflammatory phase, a proliferative phase of new tissue and matrix formation, and a remodelling phase). However, pressure ulcers often fail to progress past the inflammatory stage. Current practice for treating pressure ulcers includes treating the underlying causes, debridement to remove necrotic tissues and contaminated tissues, dressings to provide a moist wound environment and to manage exudates, devices and frequent turning of patients to provide pressure relief, topical applications of biologic agents, and nutritional support to correct nutritional deficiencies. A variety of adjunctive physical therapies are also in use.

METHODS

Health technology assessment databases and medical databases were searched from 1996 (Medline), 1980 (EMBASE), and 1982 (CINAHL) systematically up to March 2008 to identify randomized controlled trials (RCTs) on the following treatments of pressure ulcers: cleansing, debridement, dressings, biological therapies, pressure-relieving devices, physical therapies, nutritional therapies, and multidisciplinary wound care teams. Full literature search strategies are reported in appendix 1. English-language studies in previous systematic reviews and studies published since the last systematic review were included if they had more than 10 subjects, were randomized, and provided objective outcome measures on the healing of pressure ulcers. In the absence of RCTs, studies of the highest level of evidence available were included. Studies on wounds other than pressure ulcers and on surgical treatment of pressure ulcers were excluded. A total of 18 systematic reviews, 104 RCTs, and 4 observational studies were included in this review. Data were extracted from studies using standardized forms. The quality of individual studies was assessed based on adequacy of randomization, concealment of treatment allocation, comparability of groups, blinded assessment, and intention-to-treat analysis. Meta-analysis to estimate the relative risk (RR) or weighted mean difference (WMD) for measures of healing was performed when appropriate. A descriptive synthesis was provided where pooled analysis was not appropriate or not feasible. The quality of the overall evidence on each intervention was assessed using the grading of recommendations assessment, development, and evaluation (GRADE) criteria.

RESULTS

Findings from the analysis of the included studies are summarized below: CLEANSING: There is no good trial evidence to support the use of any particular wound cleansing solution or technique for pressure ulcers. DEBRIDEMENT: There was no evidence that debridement using collagenase, dextranomer, cadexomer iodine, or maggots significantly improved complete healing compared with placebo.There were no statistically significant differences between enzymatic or mechanical debridement agents with the following exceptions:Papain urea resulted in better debridement than collagenase.Calcium alginate resulted in a greater reduction in ulcer size compared to dextranomer.Adding streptokinase/streptodornase to hydrogel resulted in faster debridement.Maggot debridement resulted in more complete debridement than conventional treatment.There is limited evidence on the healing effects of debridement devices. DRESSINGS: Hydrocolloid dressing was associated with almost three-times more complete healing compared with saline gauze. There is evidence that hydrogel and hydropolymer may be associated with 50% to 70% more complete healing of pressure ulcers than hydrocolloid dressing.No statistically significant differences in complete healing were detected among other modern dressings.There is evidence that polyurethane foam dressings and hydrocellular dressings are more absorbent and easier to remove than hydrocolloid dressings in ulcers with moderate to high exudates.In deeper ulcers (stage III and IV), the use of alginate with hydrocolloid resulted in significantly greater reduction in the size of the ulcers compared to hydrocolloid alone.Studies on sustained silver-releasing dressing demonstrated a tendency for reducing the risk of infection and promoting faster healing, but the sample sizes were too small for statistical analysis or for drawing conclusions. BIOLOGICAL THERAPIES: The efficacy of platelet-derived growth factors (PDGFs), fibroblast growth factor, and granulocyte-macrophage colony stimulating factor in improving complete healing of chronic pressure ulcers has not been established.Presently only Regranex, a recombinant PDGF, has been approved by Health Canada and only for treatment of diabetic ulcers in the lower extremities.A March 2008 US Food and Drug Administration (FDA) communication reported increased deaths from cancers in people given three or more prescriptions for Regranex.Limited low-quality evidence on skin matrix and engineered skin equivalent suggests a potential role for these products in healing refractory advanced chronic pressure ulcers, but the evidence is insufficient to draw a conclusion. ADJUNCTIVE PHYSICAL THERAPY: There is evidence that electrical stimulation may result in a significantly greater reduction in the surface area and more complete healing of stage II to IV ulcers compared with sham therapy. No conclusion on the efficacy of electrotherapy can be drawn because of significant statistical heterogeneity, small sample sizes, and methodological flaws.The efficacy of other adjunctive physical therapies [electromagnetic therapy, low-level laser (LLL) therapy, ultrasound therapy, ultraviolet light therapy, and negative pressure therapy] in improving complete closure of pressure ulcers has not been established. NUTRITION THERAPY: Supplementation with 15 grams of hydrolyzed protein 3 times daily did not affect complete healing but resulted in a 2-fold improvement in Pressure Ulcer Scale for Healing (PUSH) score compared with placebo.Supplementation with 200 mg of zinc three times per day did not have any significant impact on the healing of pressure ulcers compared with a placebo.Supplementation of 500 mg ascorbic acid twice daily was associated with a significantly greater decrease in the size of the ulcer compared with a placebo but did not have any significant impact on healing when compared with supplementation of 10 mg ascorbic acid three times daily.A very high protein tube feeding (25% of energy as protein) resulted in a greater reduction in ulcer area in institutionalized tube-fed patients compared with a high protein tube feeding (16% of energy as protein).Multinutrient supplements that contain zinc, arginine, and vitamin C were associated with a greater reduction in the area of the ulcers compared with standard hospital diet or to a standard supplement without zinc, arginine, or vitamin C.Firm conclusions cannot be drawn because of methodological flaws and small sample sizes. MULTIDISCIPLINARY WOUND CARE TEAMS: The only RCT suggests that multidisciplinary wound care teams may significantly improve healing in the acute care setting in 8 weeks and may significantly shorten the length of hospitalization. However, since only an abstract is available, study biases cannot be assessed and no conclusions can be drawn on the quality of this evidence.

OBJECTIVE

The family of acyl-CoA synthetase enzymes (ACSL) activates fatty acids within cells to generate long chain fatty acyl CoA (FACoA). The differing metabolic fates of FACoAs such as incorporation into neutral lipids, phospholipids, and oxidation pathways are differentially regulated by the ACSL isoforms. In vitro studies have suggested a role for ACSL5 in triglyceride synthesis; however, we have limited understanding of the in vivo actions of this ACSL isoform.

METHODS

To elucidate the in vivo actions of ACSL5 we generated a line of mice in which ACSL5 expression was ablated in all tissues (ACSL5 (-/-) ).

RESULTS

Ablation of ACSL5 reduced ACSL activity by ∼80% in jejunal mucosa, ∼50% in liver, and ∼37% in brown adipose tissue lysates. Body composition studies revealed that ACSL5 (-/-) , as compared to control ACSL5 (loxP/loxP) , mice had significantly reduced fat mass and adipose fat pad weights. Indirect calorimetry studies demonstrated that ACSL5 (-/-) had increased metabolic rates, and in the dark phase, increased respiratory quotient. In ACSL5 (-/-) mice, fasting glucose and serum triglyceride were reduced; and insulin sensitivity was improved during an insulin tolerance test. Both hepatic mRNA (∼<em>16</em>-fold) and serum levels of <em>fibroblast</em> <em>growth</em> <em>factor</em> 21 (FGF21) (∼13-fold) were increased in ACSL5 (-/-) as compared to ACSL5 (loxP/loxP) . Consistent with increased FGF21 serum levels, uncoupling protein-1 gene (Ucp1) and PPAR-gamma coactivator 1-alpha gene (Pgc1α) transcript levels were increased in gonadal adipose tissue. To further evaluate ACSL5 function in intestine, mice were gavaged with an olive oil bolus; and the rate of triglyceride appearance in serum was found to be delayed in ACSL5 (-/-) mice as compared to control mice.

CONCLUSIONS

In summary, ACSL5 (-/-) mice have increased hepatic and serum FGF21 levels, reduced adiposity, improved insulin sensitivity, increased energy expenditure and delayed triglyceride absorption. These studies suggest that ACSL5 is an important regulator of whole-body energy metabolism and ablation of ACSL5 may antagonize the development of obesity and insulin resistance.

We recently demonstrated that alpha(v)beta(3) integrins are involved in the mechanisms of angiotensin II (Ang II)-induced DNA synthesis and collagen gel contractions in rat cardiac <em>fibroblasts</em> (CFBs), cellular mechanisms that are relevant for cardiac remodeling. The aim of the present study was to elucidate the effect of Ang II and other <em>growth</em> <em>factors</em> on the regulation of the alpha(v)beta(3) integrins in <em>fibroblasts</em> from neonatal rat hearts. The alpha(v)beta(3) integrin receptor expression was significantly increased (P<0.05) at the mRNA level after treatment with Ang II, transforming <em>growth</em> <em>factor</em>-beta(1) (TGF-beta(1)), and platelet-derived <em>growth</em> <em>factor</em> (PDGF) for 8 and <em>16</em> hours. The surface expression of the alpha(v) and beta(3) integrin subunits was elevated after 32 and 48 hours (P<0.05) as determined with flow cytometry. To investigate <em>fibroblast</em> motility, we performed chemotaxis experiments with transwell chambers. Ang II was chemotactic for CFBs, as tested with checkerboard experiments. The chemotactic effect was concentration dependent and was completely blocked by Ang II type 1 receptor blockers but not by Ang II type 2 receptor blocker PD 123319. Ang II- and PDGF-BB-mediated chemotaxis could be significantly inhibited by RGD peptides and the blocking antibodies against alpha(v)beta(3) integrin (both P<0.01). Adhesion of CFBs to vitronectin was partially inhibited by an antibody to alpha(v)beta(3) integrin but was mainly mediated by an alpha(v)beta(5) integrin. Relevant in vivo expression of alpha(v)beta(3) integrin by CFBs was confirmed with in situ hybridization with probes for alpha(v) and beta(3) mRNA in rat hearts. The present study demonstrates that the expression of alpha(v)beta(3) integrin is augmented by Ang II, PDGF, and TGF-beta(1) in neonatal CFBs. Furthermore, this integrin is involved in the chemotaxis, motility, and adhesion of CFBs. The present findings support the current concept that integrins participate in the control of <em>fibroblast</em> behavior during cardiac remodeling mechanisms.

In situ hybridization was used to document the distribution of mRNA encoding acidic and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF and bFGF) in the rat cochlea from embryonic day (E) <em>16</em> to postnatal day (P)>> 60. bFGF mRNA was not detected in the cochlea at any age. In the adult, aFGF mRNA was strongly expressed in spiral ganglion (SG) neurons, and this expression increased from base to apex. The stria vascularis (SV) and spiral prominence (SP) showed lesser expression which was equal in all turns. Developmentally, low level expression of aFGF mRNA was first seen in the SG at E-20, and remained low until P-4. Expression increased from P-6 to P-14, when adult levels were reached. aFGF mRNA was also observed in the developing hair cells of all turns at E-20. This expression increased after birth but disappeared after P-6. Expression in the SV and SP was first noted at E-20 and reached adult levels by P-<em>16</em> and P-10, respectively. High levels of aFGF mRNA in the adult SG suggest that aFGF is important for the maintenance of SG neuron function and structure. aFGF in hair cells during the first postnatal week may be involved in the establishment of cochlear innervation.

To clarify the effect of recombinant human basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) on the osteoinductive activity of recombinant human bone morphogenetic protein-2 (BMP-2) in vivo, different amounts of FGF-2 (0, <em>16</em>, 80 and 400 ng, and 2, 10 and 50 micro g: n=10 in each group), BMP-2 (2 micro g) and type I collagen as a carrier were mixed and implanted into rat calf muscles. Three weeks after implantation, compared with the controls, the radiopaque shadows of the implants were increased in the <em>16</em>, 80 and 400 ng FGF-2-treated groups, but decreased in the 2, 10 and 50 micro g FGF-2-treated groups. In addition, alkaline phosphatase activity was increased in the <em>16</em>, 80 and 400 ng FGF-2-treated groups but decreased in the 50 micro g FGF-2-treated group. Histological examination revealed increased bone formation in the <em>16</em>, 80 and 400 ng FGF-2-treated groups. These results show that combined treatment with FGF-2 and BMP-2 has a biphasic effect on osteoinductive activity, i.e. it increases with low doses of FGF-2 and decreases with high doses of FGF-2.

OBJECTIVE

The initiation, progression, and maintenance of pancreatic ductal adenocarcinoma (PDAC) results from the interplay of genetic and epigenetic events. While the genetic alterations of PDAC have been well characterized, epigenetic pathways regulating PDAC remain, for the most part, elusive. The goal of this study was to identify novel epigenetic regulators contributing to the biology of PDAC.

METHODS

In vivo pooled shRNA screens targeting 118 epigenetic proteins were performed in two orthotopic PDAC xenograft models. Candidate genes were characterized in 19 human PDAC cell lines, heterotopic xenograft tumor models, and a genetically engineered mouse (GEM) model of PDAC. Gene expression, IHC, and immunoprecipitation experiments were performed to analyze the pathways by which candidate genes contribute to PDAC.

RESULTS

In vivo shRNA screens identified BRD2 and BRD3, members of the BET family of chromatin adaptors, as key regulators of PDAC tumor growth. Pharmacologic inhibition of BET bromodomains enhanced survival in a PDAC GEM model and inhibited growth of human-derived xenograft tumors. BET proteins contribute to PDAC cell growth through direct interaction with members of the GLI family of transcription factors and modulating their activity. Within cancer cells, BET bromodomain inhibition results in downregulation of SHH, a key mediator of the tumor microenvironment and canonical activator of GLI. Consistent with this, inhibition of BET bromodomains decreases cancer-associated fibroblast content of tumors in both GEM and xenograft tumor models.

CONCLUSIONS

Therapeutic inhibition of BET proteins offers a novel mechanism to target both the neoplastic and stromal components of PDAC. Clin Cancer Res; 22(16); 4259-70. ©2016 AACR.