Migration of keratinocytes from the wound edge is thought to be one of the critical features of reepithelialization. A quantitative migration assay was carried out using normal human keratinocytes. Keratinocytes, seeded on 12 well plates, were grown in serum free, keratinocyte <em>growth</em> medium (KGM, Curabo Co) with 0.08 mM Ca2+. The medium was switched from KGM to keratinocyte basal medium (KBM) 6 h prior to the wounding. Half of the plate's confluent monolayer of keratinocytes was removed with razor blade, and the remaining keratinocytes were incubated in KBM for <em>16</em> hrs in the presence of indicated <em>growth</em> <em>factors</em>. After incubation, the cells were fixed and counted at 100 magnification. Migration was quantitated by counting the number of cells in ten successive 125-microns zones. Transforming <em>growth</em> <em>factor</em> alpha (TGF-alpha), acidic and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF, bFGF), keratinocyte <em>growth</em> <em>factor</em> (KGF), hepatocyte <em>growth</em> <em>factor</em> (HGF), and insulin-like <em>growth</em> <em>factor</em>-I (IGF-I) stimulated the migration of keratinocytes, while TGF-beta suppressed it.

In colorectal cancer, cyclooxygenase-2 (COX-2) overexpression in stromal cells induces angiogenesis through EP2 prostaglandin E2 receptor signaling. Cytoplasmic phospholipase A2 (PLA2) alpha preferentially hydrolyses arachidonic acid, which is the limiting substrate for prostaglandin production, from membrane phospholipids. We therefore investigated a possible relationship between cytoplasmic PLA2 and COX-2 overexpression in stromal cells, angiogenesis and microsatellite instability in 48 human colorectal adenocarcinomas. Cytoplasmic PLA2 and COX-2 expression in stromal cells and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) expression in tumor cells were evaluated by immunohistochemistry. Microvessel density was assessed in 10 x 400 fields after CD31 staining. Microsatellite instability was evaluated by PCR and immunohistochemistry. A total of <em>16</em> tumors had microsatellite instability. We found an overexpression of cytoplasmic PLA2 in superficial stromal cells. These cells corresponded to <em>fibroblasts</em> and myo<em>fibroblasts</em>. There was an association between the number of cytoplasmic PLA2 and COX-2-expressing cells (P=0.006). Cytoplasmic PLA2-positive stromal cells usually also expressed COX-2. A high number of cytoplasmic PLA2-positive stromal cells was correlated with a high microvessel density (P=0.002), a strong VEGF (P=0.01) and the absence of microsatellite instability (P=0.001). The coordinate overexpression of cytoplasmic PLA2 and COX-2 in stromal cells could lead to an important prostaglandin production. These results suggest that cytoplasmic PLA2 overexpression in these cells regulates COX-induced angiogenesis probably by providing arachidonic acid, which is the limiting <em>factor</em> for prostaglandin production. The lower number of cytoplasmic PLA2-positive stromal cells in carcinomas with microsatellite instability could be related to their lower microvessel density and VEGF expression.

We report the primary structure of MG-<em>16</em>0, a <em>16</em>0 kDa membrane sialoglycoprotein residing in the medial cisternae of the Golgi apparatus of rat neurons, pheochromocytoma (PC-12), and several other cells. The cDNA encodes a polypeptide of 1,171 amino acids with an M(r) of 133,403. An intralumenal cleavable signal peptide is followed by a Pro-Gln-rich segment and <em>16</em> contiguous, approx. 60-residue-long, regularly spaced cysteine-rich segments showing sequence identities ranging from 15 to 35%. The lumenal domain is followed by a single membrane spanning domain and a short carboxy-terminal cytoplasmic tail. The protein contains 5 potential NXT glycosylation sites. The sequence of MG-<em>16</em>0 shows no homologies with enzymes and other membrane proteins of the Golgi apparatus. MG-<em>16</em>0 displays a so far unique feature for a membrane protein of the Golgi apparatus: namely, an upstream, open reading frame (uORF), encoding 58 amino acids, located in front of the major open reading frame (ORF). Most vertebrate mRNAs containing uORF or AUG codons in front of the major ORF encode <em>growth</em> <em>factors</em> and cell surface receptors (Geballe and Morris 1994). In that regard a 90% identity between the primary structure of MG-<em>16</em>0 and a receptor for acidic and basic <em>fibroblast</em> <em>growth</em> <em>factors</em> (CFR), isolated from chicken embryos (Burrus et. al., 1992), may be relevant. Immunoreactivity for MG-<em>16</em>0 has been detected in the Golgi apparatus of neural and other cells of 2-day-old chicken embryos and adult chicken; furthermore, recombinant human basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) binds MG-<em>16</em>0 purified from rat brain. MG-<em>16</em>0 shows no sequence similarity with members of the family of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptors (FGFR) involved in signal transduction. These findings are consistent with the hypothesis that MG-<em>16</em>0 is involved in the traffic and processing of endogenous or autocrine FGFs. This is the first example of an intrinsic membrane protein of the Golgi apparatus which binds a <em>growth</em> <em>factor</em> and may be involved in its regulation.

BACKGROUND

Angiogenesis appears to be an important event in the pathophysiology of endometriosis (EM) and adenomyosis. Two angiogenic factors, fibroblast growth factor (FGF) 1 and 2, play a central role in the initiation of angiogenesis. We investigated whether FGF1 -1385A/G and FGF2 754C/G polymorphisms are associated with a risk of developing EM and adenomyosis.

METHODS

Genotypes were analyzed by the PCR-restriction fragment length polymorphism method in two groups of women, of Han ethnicity in north China, aged 16-55 years: (1) 421 EM patients and 421 controls; (2) 269 adenomyosis patients and 269 controls.

RESULTS

There was no difference in genotype distribution of the FGF1 -1385A/G polymorphism between adenomyosis cases and controls (P>> 0.05), but the frequency of the A allele in EM patients was lower than that in controls (P = 0.013). Genotype and allele frequencies of the FGF2 754C/C polymorphism were significantly different in both EM and adenomyosis cases versus control groups. Compared with C/C homozygotes, the G allele (C/G + G/G) was associated with a decreased susceptibility to developing EM [odds ratio (OR) = 0.575, 95% confidence interval (CI) = 0.387-0.854] and adenomyosis (OR = 0.577, 95% CI = 0.367-0.906). Combined genotype analysis of both polymorphisms also showed differences between cases versus controls (all P < 0.001).

CONCLUSIONS

Our study shows for the first time that the FGF2 754C/G polymorphism may be associated with a risk of developing EM and adenomyosis in north Chinese women. Carriers of the G allele in the FGF2 gene appear to be protected from these gynecological diseases. Further studies in other populations, and of other candidate genes, are now warranted.

Aromatase (estrogen synthetase) is expressed in breast cancer tissue, and in situ expression of the enzyme stimulates breast cancer <em>growth</em>. Promoter I.3 is one of the major promoters that control the expression of aromatase in breast cancer tissue. Using the yeast one-hybrid approach to screen a human breast tissue hybrid cDNA expression library, we found that the zinc-finger transcriptional <em>factor</em> Snail (SnaH) interacted with a regulatory region near promoter I.3 of the human aromatase gene. DNA mobility shift assays and mutation analyses using recombinant SnaH protein expressed in Escherichia coli have revealed that this protein interacts with a segment, 5'-CTGATGAAGT-3', which is between 66 and 76 bp upstream from the transcriptional start site of promoter I.3. Using mammalian cell transfection experiments, SnaH was found to act as a repressor of promoter I.3 activity. Site-directed mutagenesis experiments have revealed that the NH2-terminal SNAG domain is important for the repressor activity of SnaH. To demonstrate the inhibitory activity against aromatase expression, a stable SnaH-expressing MDA-MB-231 breast cancer cell line was generated, and the aromatase RNA messages in the SnaH-transfected cell line were found to be 30% of those in the vector-transfected cell line. Reverse transcription-PCR analysis on RNAs isolated from 12 cell lines has confirmed that SnaH is expressed at a higher level in normal breast epithelial cell and stromal <em>fibroblast</em> cell lines than in breast cancer cell lines. In addition, SnaH mRNA was detected in only <em>16</em> of 55 breast cancer specimens. On the other hand, aromatase mRNA was detected in 54 of the 55 specimens. Our results indicate that SnaH acts as a repressor that down-regulates the expression of aromatase in normal breast tissue by suppressing the function of promoter I.3. A reduction of the expression of SnaH in breast cancer tissue further suggests a cancer-protective role for this protein in normal breast tissue.

The fate of iodinated basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) after its binding to cultured astrocytes and hippocampal neurons was studied. Autoradiography after light and electron microscopy establishes that, if cells are returned to 37 degrees C, the 125I-basic FGF bound internalizes into vesicles in the cytoplasm, localizes to the perinuclear cytoplasm, and is translocated to chromatin structures of the nucleus. The radiolabeled protein is long-lived, a finding confirmed by biochemical analyses. Polyacrylamide gel electrophoresis and autoradiography of both hippocampal neurons and astrocyte extracts reveal that these cells internalize 125I-basic FGF and then metabolize it to three major heparin-binding peptides with molecular weights of 15.5, 9, and 4 kDa. These peptides are initially detected <em>16</em> hr after binding to neurons and 4 hr after binding to astrocytes but are still detectable 48 and <em>16</em> hr, respectively, after initial binding (though present at lower levels). Immunoprecipitation with sequence-specific antisera to basic FGF reveals that the 15.5-kDa fragment is generated by cleavage at the carboxyl terminus, that the 9-kDa peptide contains the sequences between residues 30 and 87, and the 4-kDa peptide is a C-terminus fragment containing the sequence of basic FGF(106-120) but without basic FGF(139-146) immunoreactivity. The internalization of basic FGF is required for this processing; the treatment of cells with trypsin and 2 M NaCl at different times after binding can only prevent the metabolism of basic FGF if it is performed immediately after binding. Similarly, WGA, which inhibits basic FGF binding to its high-affinity receptor, prevents the metabolism of basic FGF. The possible significance of a metabolic pathway that is responsible for the processing of basic FGF after its internalization by cells in the CNS is discussed in light of its potential function as a neurotrophic <em>factor</em>.

During endochondral bone formation, cartilage cells show increased matrix synthesis and rapid proliferation. We found that cartilage matrix contains at least two types of heparin binding <em>growth</em>-promoting components. One, with a higher affinity to heparin, was identified as chondromodulin I (Hiraki, Y., Tanaka, H., Inoue, H. , Kondo, J., Kamizono, A., and Suzuki, F. (1991) Biochem. Biophys. Res. Commun. 175, 871-977). In this study, we isolated a novel <em>growth</em>-promoting component, chondromodulin II, which has a lower heparin affinity, from the dissociative extracts of fetal bovine epiphyseal cartilage. Chondromodulin II stimulated the proteoglycan synthesis in rabbit cultured <em>growth</em> plate chondrocytes, an expression of the differentiated phenotype of chondrocytes. It also stimulated DNA synthesis in chondrocytes in both the absence and the presence of <em>fibroblast</em> <em>growth</em> <em>factor</em>-2. The apparent molecular mass of chondromodulin II on SDS-polyacrylamide gel electrophoresis was <em>16</em> kDa. Its complete amino acid sequence was determined by overlapping sequences of the peptides released by endopeptidase digestion and CNBr cleavage. Chondromodulin II consists of 133 amino acids (calculated Mr = 14,548). The sequence was unique but homologous to the repeats 1 and 2 of the deduced amino acid sequence of the chicken mim-1 gene, which is specifically transactivated by the v-Myb oncogene product in promyelocytes. We also found a minor component with a higher heparin affinity, chondromodulin III, in cartilage extracts. Chondromodulin III stimulated DNA synthesis in chondrocytes in vitro, and its N-terminal sequence was identical with ribosomal protein L31 lacking the N-terminal three amino acids. These findings suggest that the <em>growth</em> and differentiation of chondrocytes are regulated by multiple components in the cartilage matrix.

Several investigators have postulated that soluble <em>growth</em> <em>factors</em> are involved in the early development of the pancreas. In many tissues in which soluble <em>factors</em> are implicated in development, these <em>factors</em> act on their target cells through tyrosine kinase receptors. Because we had some preliminary evidence that <em>fibroblast</em> <em>growth</em> <em>factor</em> receptors (FGFRs) were expressed in the early pancreas, we investigated the effect of <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) during embryonic pancreatic development. For that purpose, we first studied the distribution and the functionality of FGFRs during pancreatic organogenesis. FGFR1 and FGFR4 were shown to be expressed at a high level during early pancreatic development before embryonic day <em>16</em>, their levels of expression decreasing thereafter. The functionality of FGFR was studied next. It was demonstrated in vitro that both FGF1 and FGF2 induce the expression of NGFI-A mRNA, a useful indicator of functional <em>growth</em> <em>factor</em>-signaling pathways. Finally, the effect of FGF2 on embryonic pancreatic epithelial cell proliferation was studied. It was shown that FGF2 induces the proliferation of pancreatic epithelial cells during embryonic life. Taken together, these data strongly suggest that FGFs are implicated in pancreatic development during embryonic life.

The HIV-1 trans-activation responsive element (TAR) RNA stem-loop interacts with the HIV trans-activator protein Tat and other cellular <em>factors</em> to stimulate transcriptional elongation from the viral long terminal repeat (LTR). Inhibitors of these interactions block full-length transcription and, hence, would potentially inhibit HIV replication. We have studied structure-activity relationships in inhibition of trans-activation by steric block 2'-O-methyl (OMe) oligonucleotides chimeras (mixmers) containing locked nucleic acid (LNA) units. Inhibition was measured both in Tat-dependent in vitro transcription from an HIV-1 DNA template directed by HeLa cell nuclear extract and in a robust HeLa cell reporter assay that involves use of stably integrated plasmids to express firefly luciferase Tat dependently and Renilla luciferase Tat-independently. OMe oligonucleotides with optimally 40%-50% LNA units and a minimum of 12 residues in length were active in the cellular assay when delivered with cationic gemini surfactant GS11 at 50% inhibitory concentrations of 230 +/- 40 nM, whereas activity in the in vitro transcription assay was observed down to 9 residues. No cellular activity was observed for OMe oligonucleotides of 12 or <em>16</em> residues, which was shown to be due to poor cellular uptake. Both 12-mer mixmers containing alpha -L-LNA or 2'-thio-LNA (S-LNA) were also active in in vitro transcription and the former in cellular reporter inhibition assays, demonstrating that the property of promotion of cellular uptake by LNA is not due to specific sugar conformational effects. Covalent conjugates of OMe/LNA chimeras with Kaposi-<em>fibroblast</em> <em>growth</em> <em>factor</em> (K-FGF) or Transportan peptides failed to enter HeLa cells without a delivery agent but were fully active when delivered by cationic gemini surfactant, showing that in principle, peptide conjugation does not interfere with cellular activity. Thus, OMe/LNA mixmers are powerful reagents for use as steric block inhibitors of gene expression regulated by protein-RNA interactions within HeLa cell nuclei.

Incubation of Swiss 3T3 cells with [2-3H]adenine, as in other cell types, reveals the ADP-ribosylation of GRP78 (the 78-kDa glucose-regulated protein, also known as BiP, the immunoglobulin heavy chain-binding protein), a resident endoplasmic reticulum protein that assists in the processing of proteins destined for secretion or cell surface expression. Here we show that Pasteurella multocida toxin, a potent <em>growth</em> <em>factor</em> for cultured <em>fibroblasts</em>, decreased the ADP-ribosylation of GRP78/BiP to <em>16</em> +/- 6% of the control value (n = 23). The action of the toxin occurred after a lag period, was blocked by lysosomotrophic agents, and potentiated by increased incubation time (ED50 4 ng/ml and 1 ng/ml in 4 and 8 h, respectively), thus indicating that the toxin enters the cells to act. Bombesin and platelet-derived <em>growth</em> <em>factor</em> (PDGF) similarly decreased the ADP-ribosylation of GRP78/BiP (ED50 0.5 nM and 2.5 ng/ml, respectively) but acted more rapidly than the toxin. Signaling pathways activated by the toxin, bombesin, and PDGF had effects on the ADP-ribosylation of GRP78/BiP. Thus, activation of protein kinase C alone by phorbol 12,13-dibutyrate was partially effective, and down-regulation of protein kinase C attenuated but did not block the action of the toxin, bombesin, and PDGF. Agents that mobilize Ca2+ from the endoplasmic reticulum (A23187, ionomycin, and thapsigargin) caused a decrease in the ADP-ribosylation of GRP78/BiP that was similar in magnitude to that achieved by the toxin, bombesin, and PDGF, implicating a role for inositol 1,4,5-trisphosphate-mediated Ca2+ mobilization in the action of the mitogenic agents. The <em>growth</em> <em>factor</em>-induced decrease in the ADP-ribosylation of GRP78/BiP may represent its conversion from an inactive to an active state.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is a potent endothelial cell mitogen found in a variety of normal and tumor tissues. Basic FGF lacks a classical signal sequence, and it is not clear how it is released from cells. bFGF or bFGF-like activity has not been previously demonstrated in plasma. In an earlier study we showed increased mitogenic activity for parathyroid-derived epithelial and mesenchymal cells in plasma of subjects with familial multiple endocrine neoplasia type 1 (FMEN1). In the present study we examined the <em>growth</em>-promoting activity of normal and FMEN1 plasmas [applied to heparin-Sepharose (HS) columns] in parathyroid-derived cloned endothelial cells. FMEN1 plasma HS-adsorbed activity exceeded normal plasma HS-adsorbed activity in 6 of 8 FMEN1 plasma samples. Peak (FMEN1 plasma) HS-adsorbed activity eluted with 0.1-0.3 M NaCl, was completely neutralized by specific antibodies against bFGF, and had an apparent mol wt of 110 kD. Active fractions from FMEN1 plasma prepared by gel filtration in 7 M urea displayed apparent mol wt of about 14-<em>16</em> kD and showed increased apparent affinity for HS; recovered activity appeared principally in the 3.0-M NaCl eluate. Using a sensitive two-site immunoradiometric assay for bFGF we found 0.4 ng/mL bFGF-like immunoreactivity in the highly purified 3.0-M NaCl eluate from a HS column to which the active components from gel filtration of FMEN1 plasma in 7 M urea were applied. These results imply that bFGF or closely related <em>factors</em> circulate in FMEN1.

Tissue-specific alternative splicing in the second half of Ig-like domain 3 (D3) of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptors 1-3 (FGFR1 to -3) generates epithelial FGFR1b-FGFR3b and mesenchymal FGFR1c-FGFR3c splice isoforms. This splicing event establishes a selectivity filter to restrict the ligand binding specificity of FGFRb and FGFRc isoforms to mesenchymally and epithelially derived <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs), respectively. FGF1 is termed the "universal FGFR ligand" because it overrides this specificity barrier. To elucidate the molecular basis for FGF1 cross-reactivity with the "b" and "c" splice isoforms of FGFRs, we determined the first crystal structure of FGF1 in complex with an FGFRb isoform, FGFR2b, at 2.1 Å resolution. Comparison of the FGF1-FGFR2b structure with the three previously published FGF1-FGFRc structures reveals that plasticity in the interactions of the N-terminal region of FGF1 with FGFR D3 is the main determinant of FGF1 cross-reactivity with both isoforms of FGFRs. In support of our structural data, we demonstrate that substitution of three N-terminal residues (Gly-19, His-25, and Phe-26) of FGF2 (a ligand that does not bind FGFR2b) for the corresponding residues of FGF1 (Phe-<em>16</em>, Asn-22, and Tyr-23) enables the FGF2 triple mutant to bind and activate FGFR2b. These findings taken together with our previous structural data on receptor binding specificity of FGF2, FGF8, and FGF10 conclusively show that sequence divergence at the N termini of FGFs is the primary regulator of the receptor binding specificity and promiscuity of FGFs.

Clinical efficacy of stem cells for nerve repair is likely to be influenced by issues including donor age and in vitro expansion time. We isolated human mesenchymal stem cells (MSC) from bone marrow of young (<em>16</em>-18 years) and old (67-75 years) donors and analyzed their capacity to differentiate and promote neurite out<em>growth</em> from dorsal root ganglia (DRG) neurons. Treatment of MSC with <em>growth</em> <em>factors</em> (forskolin, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, platelet derived <em>growth</em> <em>factor</em>-AA and glial <em>growth</em> <em>factor</em>-2) induced protein expression of the glial cell marker S100 in cultures from young but not old donors. MSC expressed various neurotrophic <em>factor</em> mRNA transcripts. <em>Growth</em> <em>factor</em> treatment enhanced the levels of BDNF and VEGF transcripts with corresponding increases in protein release in both donor cell groups. MSC in co-culture with DRG neurons significantly enhanced total neurite length which, in the case of young but not old donors, was further potentiated by treatment of the MSC with the <em>growth</em> <em>factors</em>. Stem cells from young donors maintained their proliferation rate over a time course of 9 weeks whereas those from the old donors showed increased population doubling times. MSC from young donors, differentiated with <em>growth</em> <em>factors</em> after long-term culture, maintained their ability to enhance neurite out<em>growth</em> of DRG. Therefore, MSC isolated from young donors are likely to be a favourable cell source for nerve repair.

BACKGROUND

Although the systemic administration of deferoxamine (DFO) is protective in experimental models of normal ischemic flap and diabetic wound, its effect on diabetic flap ischemia using a local injection remains unknown.

OBJECTIVE

To explore the feasibility of local injection of DFO to improve the survival of ischemic random skin flaps in streptozotocin (STZ)-induced diabetic mice.

METHODS

Ischemic random skin flaps were made in 125 mice. Animals were divided into the DFO-treated (n = 20), PBS-treated (n = <em>16</em>) and untreated (n = <em>16</em>) groups. Surviving area, vessel density, and expression of vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and hypoxia-inducible <em>factor</em>-1α (HIF-1α) were evaluated on the seventh day after local injection.

RESULTS

The viability of DFO-treated flap was significantly enhanced, with increased regional blood perfusion and capillary density compared with those in the two control groups. Fluorescence-activated cell sorting (FACS) analysis demonstrated a marked increase in systemic Flk-1+/CD11b- endothelial progenitor cells (EPCs) in DFO-treated mice. Furthermore, the expression of VEGF and HIF-1α was increased not only in diabetic flap tissue, but also in dermal fibroblasts cultured under hyperglycemic and hypoxic conditions.

CONCLUSIONS

Local injection of DFO could exert preventive effects against skin flap necrosis in STZ-induced diabetic mice by elevating the expression of HIF-1α and VEGF, increased EPC mobilization, which all contributed to promote ischemic diabetic flap survival.

OBJECTIVE

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor-inducible molecule 14 (Fn14) are a ligand-receptor pair frequently overexpressed in solid tumors.

UNASSIGNED

Fn14 signaling regulates multiple oncogenic processes through MAPK, AKT, and NFκB pathway activation. A phase I study of RG7212, a humanized anti-TWEAK IgG1κ monoclonal antibody, was conducted in patients with advanced solid tumors expressing Fn14.

METHODS

Dose escalations, over a 200- to 7,200-mg range, were performed with patients enrolled in weekly (QW), bi-weekly (Q2W), or every-three-week (Q3W) schedules. Primary objectives included determination of dose and safety profile. Secondary endpoints included assessments related to inhibition of

UNASSIGNED

Fn14 signaling, tumor proliferation, tumor immune cell infiltration, and pharmacokinetics.

RESULTS

In 192 treatment cycles administered to 54 patients, RG7212 was well-tolerated with no dose-limiting toxicities observed. More than 95% of related adverse events were limited to grade 1/2. Pharmacokinetics were dose proportional for all cohorts, with a t1/2 of 11 to 12 days. Pharmacodynamic changes included clearance of free and total TWEAK ligand and reductions in tumor Ki-67 and TRAF1. A patient with BRAF wild-type melanoma who received 36 weeks of RG7212 therapy had tumor regression and pharmacodynamic changes consistent with antitumor effects. Fifteen patients (28%) received 16 or more weeks of RG7212 treatment.

CONCLUSIONS

RG7212 demonstrated excellent tolerability and favorable pharmacokinetics. Pharmacodynamic endpoints were consistent with reduced

UNASSIGNED

Fn14 signaling. Tumor regression was observed and prolonged stable disease was demonstrated in multiple heavily pretreated patients with solid tumors. These encouraging results support further study of RG7212. Clin Cancer Res; 21(2); 258-66. ©2014 AACR.

OBJECTIVE

The development of fibrotic diseases is associated with alterations in the transforming growth factor beta (TGF-beta) pathway. We have investigated the expression and activity of Smad transcription factors of the TGF-beta pathway in primary tunical fibroblasts derived from patients with Peyronie's disease and from controls.

METHODS

Primary fibroblasts were established from biopsies obtained from plaques of 16 patients with Peyronie's disease or the tunica albuginea of 8 control patients. The expression and activity of Smad transcription factors in control and TGF-beta-stimulated primary fibroblasts were investigated at the RNA and protein level by reverse transcription-polymerase chain reaction, Western blotting, and immunofluorescence.

RESULTS

RNA expression levels of Smad3 and Smad4 were significantly increased in fibroblasts from patients with Peyronie's disease. When stimulated with TGF-beta1, fibroblasts showed rapid nuclear translocation of Smad2/3, as soon as 15 min after stimulation. This effect was more pronounced and exhibited an earlier onset in fibroblasts from patients with Peyronie's disease, compared with controls. In addition, an increased nuclear retention time of Smad4 was observed in fibroblasts from patients with Peyronie's disease.

CONCLUSIONS

The expression and activity of Smad transcription factors of the TGF-beta pathway is increased in fibroblasts of patients with Peyronie's disease. Alterations in the TGF-beta pathway seem to be a pathogenetic factor in the development of Peyronie's disease.

Proprotein convertase (PC) 5/6 belongs to a family of secretory proteases involved in proprotein proteolysis. Several studies suggest a role for PC5/6 in cardiovascular disease. Because lethality at birth of mice lacking PC5/6 precluded elucidation of its function in the adult, we generated mice in which the gene of PC5/6 (pcsk5) is specifically inactivated in endothelial cells (ecKO), which are viable and do not exhibit overt abnormalities. In order to uncover the function of PC5/6 in the cardiovascular system, the effect of ecKO was studied in aging mice. In <em>16</em> to 18-month-old ecKO mice, the left ventricle (LV) mass, media cross-sectional area of aorta and coronary arteries, and media-to-lumen ratio of mesenteric arteries were decreased. The LV presented decreased diastolic function, and mesenteric arteries showed decreased stiffness. Collagen was decreased in the LV myocardial interstitium and perivascularly in coronary arteries and aorta. Cardiovascular hypotrophy likely develops with aging, since no significant changes were observed in 2-month-old ecKO mice. <em>Fibroblasts</em>, as a source of collagen in myocardium and vasculature, may play a role in the decrease in collagen deposition. <em>Fibroblasts</em> co-cultured with ecKO endothelial cells showed decreased collagen production, decreased insulin-like <em>growth</em> <em>factor</em> (IGF)-1/Akt/mTOR signaling, and enhanced autophagic activation. PC5/6 inactivation in endothelial cells results in cardiovascular hypotrophy associated with decreased collagen deposition, decreased LV diastolic function, and vascular stiffness, suggesting a trophic role of endothelial PC5/6 in the cardiovascular system, likely mediated by IGF-1/Akt/mTOR signaling and control of autophagy.

Controversial correlations between biological activity and concentration of the novel lipokine palmitoleate (9Z-hexadecenoate, <em>16</em>:1) might depend on the formation of an active <em>16</em>:1 metabolite. For its identification, we analyzed the glycerophospholipid composition of mouse Swiss 3T3 <em>fibroblasts</em> in response to <em>16</em>:1 using LC-MS/MS. <em>16</em>:1 was either supplemented to the cell culture medium or endogenously formed when cells were stimulated with insulin or <em>growth</em> <em>factors</em> as suggested by the enhanced mRNA expression of <em>16</em>:1-biosynthetic enzymes. The proportion of 1-acyl-2-<em>16</em>:1-sn-phosphatidylinositol (<em>16</em>:1-PI) was time-dependently and specifically increased relative to other glycerophospholipids under both conditions and correlated with the proliferation of fatty acid (<em>16</em>:1, palmitate, oleate, or arachidonate)-supplemented cells. Accordingly, cell proliferation was impaired by blocking <em>16</em>:1 biosynthesis using the selective stearoyl-CoA desaturase-1 inhibitor CAY10566 and restored by supplementation of <em>16</em>:1. The accumulation of <em>16</em>:1-PI occurred throughout cellular compartments and within diverse mouse cell lines (Swiss 3T3, NIH-3T3, and 3T3-L1 cells). To elucidate further whether <em>16</em>:1-PI is formed through the de novo or remodeling pathway of PI biosynthesis, phosphatidate levels and lyso-PI-acyltransferase activities were analyzed as respective markers. The proportion of <em>16</em>:1-phosphatidate was significantly increased by insulin and <em>growth</em> <em>factors</em>, whereas lyso-PI-acyltransferases showed negligible activity for <em>16</em>:1-coenzyme A. The relevance of the de novo pathway for <em>16</em>:1-PI biosynthesis is supported further by the comparable incorporation rate of deuterium-labeled <em>16</em>:1 and tritium-labeled inositol into PI for <em>growth</em> <em>factor</em>-stimulated cells. In conclusion, we identified <em>16</em>:1 or <em>16</em>:1-PI as mitogen whose biosynthesis is induced by <em>growth</em> <em>factors</em>.

The neuroprotective effects of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) on the long-term survival of axotomized retinal ganglion cells (RGCs) were studied in the frog Rana pipiens. Cell loss was quantified in different regions of the ganglion cell layer using Nissl staining and tetramethylrhodamine dextran amine backfilling. All regions of the retina showed a significant decrease (32-66%) in RGC numbers between 4 and <em>16</em> weeks after axotomy. Some cells showed morphological and biochemical signs of apoptosis. A single application of bFGF to the optic nerve stump at the time of axotomy protected many of the cells 6 weeks after the injury, but this effect was lost by 12 weeks. A second application of bFGF, 6 weeks after the injury, rescued many RGCs at 12 weeks. In contrast, single or double injections of bFGF into the eyeball had no effect on RGC survival. Axotomized RGCs were significantly enlarged and elongated after axotomy, and these morphological changes were increased by bFGF treatment. In the normal retina and optic nerve, immunocytochemical staining showed bFGF-like immunoreactivity (-LI) in the pigment epithelial layer, in the outer segments of photoreceptors, and in occasional RGCs. Strong bFGF-LI was present in Müller cells and in optic nerve astrocytes and oligodendrocytes. FGF receptor-LI was present in photoreceptors, outer plexiform layer, retinal ganglion cell axons, and Müller cells. FGF receptor-LI was also observed in optic nerve glia.

<b>Rationale:</b> Angiogenesis promotes neurological recovery after stroke and is associated with longer survival of stroke patients. Cerebral angiogenesis is tightly controlled by certain microRNAs (miRs), such as the miR-15a/<em>16</em>-1 cluster, among others. However, the function of the miR-15a/<em>16</em>-1 cluster in endothelium on post-ischemic cerebral angiogenesis is not known. <b>Objective:</b> To investigate the functional significance and molecular mechanism of endothelial miR-15a/<em>16</em>-1 cluster on angiogenesis in the ischemic brain. <b>Methods and Results:</b> Endothelial cell -selective miR-15a/<em>16</em>-1 conditional knockout (EC-miR-15a/<em>16</em>-1 cKO) mice and WT littermate controls were subjected to 1h middle cerebral artery occlusion (MCAO) followed by 28d reperfusion. Deletion of miR-15a/<em>16</em>-1 cluster in endothelium attenuates post-stroke brain infarction and atrophy, and improves the long-term sensorimotor and cognitive recovery against ischemic stroke. Endothelium-targeted deletion of the miR-15a/<em>16</em>-1 cluster also enhances post-stroke angiogenesis by promoting vascular remodeling and stimulating the generation of newly formed functional vessels, and increases the ipsilateral cerebral blood flow. Endothelial cell-selective deletion of the miR-15a/<em>16</em>-1 cluster up-regulated the protein expression of pro-angiogenic <em>factors</em> vascular endothelial <em>growth</em> <em>factor</em> (VEGFA), <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2), and their receptors VEGFR2 and FGFR1 after ischemic stroke. Consistently, lentiviral knockdown of the miR-15a/<em>16</em>-1 cluster in primary mouse or human brain microvascular endothelial cell cultures enhanced in vitro angiogenesis and up-regulated pro-angiogenic proteins expression after oxygen-glucose deprivation (OGD), whereas lentiviral overexpression of the miR-15a/<em>16</em>-1 cluster suppressed in vitro angiogenesis and down-regulated pro-angiogenic proteins expression. Mechanistically, miR-15a/<em>16</em>-1 translationally represses pro-angiogenic <em>factors</em> VEGFA, FGF2, and their receptors VEGFR2 and FGFR1, respectively, by directly binding to the complementary sequences within three prime untranslated regions (3'-UTRs) of those mRNAs. <b>Conclusions:</b> Endothelial miR-15a/<em>16</em>-1 cluster is a negative regulator for post-ischemic cerebral angiogenesis and long-term neurological recovery. Inhibition of miR-15a/<em>16</em>-1 function in cerebrovascular endothelium may be a legitimate therapeutic approach for stroke recovery.

1. Currently, there is no satis<em>factor</em>y treatment for pulmonary fibrosis. Emodin, a component in Chinese herbs, has been shown to have an antifibrotic effect on pancreatic fibrosis and liver fibrosis. In the present study, we tested the hypothesis that emodin may attenuate the development of pulmonary fibrosis. 2. Mice were randomly divided into five groups (n = <em>16</em> in each). One group was a control group; the remaining four groups were treated with intratracheal instillation of 3 mg/kg bleomycin (BLM). The following day, emodin (5, 10 or 20 mg/kg per day, p.o.) treatment was started for three of the BLM-treated groups and was continued for 21 days. The fourth BLM-treated group (and the control group) received daily 0.5% sodium carboxymethyl cellulose (placebo) by gavage over the same period. 3. Bleomycin challenge provoked severe pulmonary fibrosis, with marked increases in fibrosis fraction, hydroxyproline content and myeloperoxidase activity in lung tissue. Emodin treatment (10 and 20 mg/kg per day, p.o.) attenuated all these biochemical indices, as well as histopathological alterations induced by BLM. Furthermore, in mice injected with BLM, elevated levels of transforming <em>growth</em> <em>factor</em>-beta1, interleukin (IL)-4 and IL-13 were found in bronchoalveolar lavage fluid. These increases were significantly inhibited by 10 and 20 mg/kg per day emodin. 4. In cell culture, exposure of cells to 6.25, 12.5, 25 or 50 micromol/L emodin for 24 h decreased <em>fibroblast</em> proliferation. Treatment of cells with the same concentrations of emodin for 72 h decreased collagen production by <em>fibroblasts</em>. In addition, emodin (6.25, 12.5, 25 or 50 micromol/L) inhibited the steady state expression of alpha1 (I) procollagen and alpha2 (I) procollagen mRNA in a dose-dependent manner. 5. The results of the present study suggest that emodin may be effective in the treatment of pulmonary fibrosis.

BACKGROUND

The development of urothelial malignancy is not solely a consequence of loss of proliferation constraints but also involves loss of cellular differentiation, defined histopathologically as grade. Although tumour grade is an independent prognostic marker for urothelial carcinoma (UC), the molecular events underpinning the loss of urothelial differentiation are poorly understood.

OBJECTIVE

To examine the effect of gene alterations implicated in UC development on the ability of human urothelial cells to undergo molecular differentiation and form a functional urothelial barrier.

METHODS

Laboratory study.

METHODS

Normal human urothelial (NHU) cell cultures were transduced with recombinant retroviruses to produce stable sublines overexpressing wild-type or oncogenic mutated <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 or human telomerase reverse transcriptase (hTERT). Previously generated NHU sublines carrying dominant-negative CDK4 and p53 mutant genes or immortalised with the human papillomavirus <em>16</em> E6 oncoprotein were included.

METHODS

The activity of introduced transgenes was demonstrated by comparing phenotypes of transgene-expressing and isogenic control NHU cells. Modified and control sublines were compared for changes in generational potential (life span) and capacity to respond to differentiation-inducing signals by transcript expression of uroplakins 2 and 3. The ability to form a barrier epithelium was assessed by measuring the transepithelial electrical resistance.

CONCLUSIONS

By contrast to tumour suppressor loss of function or oncogene overactivation, hTERT overexpression alone led to life span extension and immortalisation. The hTERT immortalised cells carried no gross genomic alterations but became progressively insensitive to differentiation signals and lost the ability to form an epithelial barrier. Further characterisation of hTERT cells revealed a downregulation of p<em>16</em> cyclin-dependent kinase inhibitor expression and loss of responsiveness to peroxisome proliferator-activated receptor γ, providing mechanistic explanations for the subjugation of senescence constraints and the abrogation of differentiation capability, respectively. Although immortalised urothelial cell lines without karyotypic aberrations may be generated, such cell lines are compromised in terms of differentiation and functional capacity.

CONCLUSIONS

Overexpression of hTERT promotes development of an immortalised differentiation-insensitive urothelial cell phenotype. Although such cells offer a useful insight into the grade/stage paradigm of UC, they have limited value for investigating normal urothelial cell/tissue biology and physiology.

Cervical cancer is the most common malignancy in females worldwide. This study investigated the prevalence of the E6/E7 oncoproteins of human papillomavirus (HPV) type <em>16</em>, which are important in <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) 2- and 4-induced epithelial-mesenchymal transition (EMT) and cervical tumorigenesis. We investigated the functional interaction between HPV<em>16</em> E6/E7-transfected Cx cells (CxWJ cells) and treatment with FGF2 and 4, according to the expression of α-smooth muscle actin (α-SMA), vimentin and E-cadherin protein as well as cell <em>growth</em> and invasive ability. The results showed the upregulation of α-SMA and vimentin and the downregulation of E-cadherin protein expression in CxWJ cells. HPV<em>16</em> E6/E7 infection partially repressed proliferation, but not the invasive ability of FGF2 or FGF4 stimulation in cervical cancer cells (CxWJ cells). These data provide evidence of a functional interaction between HPV<em>16</em> E6/E7 and FGFs 2 and 4, suggesting that cooperative stimulation of HPV E6/E7 and FGFs activated in human cervical cancer cells is required to completely overcome the oncogenic function associated with the development of cervical epithelial-mesenchymal transition and tumorigenesis.

Reorganization of skin during wound healing, inflammatory disorders, or cancer <em>growth</em> is the result of expression changes of multiple genes associated with tissue morphogenesis. We wanted to identify proteins involved in skin remodeling and select those that may be targeted for agonistic or antagonist therapeutic approaches in various disease processes. Full-thickness human skin was grafted to severe combined immunodeficient mice and injected intradermally with 38 different adenoviral vectors inserted with 37 different genes coding for <em>growth</em> <em>factors</em>, cytokines, proteolytic enzymes and their inhibitors, adhesion receptors, oncogenes, and tumor suppressor genes. Responses were characterized for infiltration of inflammatory cells, vascular density, matrix formation, <em>fibroblast</em>-like cell proliferation, and epidermal hyperplasia. Of the 17 <em>growth</em> <em>factor</em> vectors, <em>16</em> induced histological changes in human skin. Members of the VEGF and angiopoietin families induced neovascularization. PDGFs and TGF-betas stimulated connective tissue formation, and the chemokines IL-8 and MCP-1 attracted inflammatory neutrophils and monocytes, respectively. The serine protease uPA induced a vascular response similar to that of VEGF. Vectors with adhesion receptors, oncogenes and tumor suppressor genes had, with few exceptions, little effects on skin architecture. The overall results suggest that adenoviral vectors can effectively remodel the architecture of human skin for studies in morphogenesis, inflammatory skin disorders, wound healing, and cancer development.