Fibulin-4 is a member of the fibulin family, a group of extracellular matrix proteins prominently expressed in medial layers of large veins and arteries. Involvement of the FBLN4 gene in cardiovascular pathology was shown in a murine model and in three patients affected with cutis laxa in association with systemic involvement. To elucidate the contribution of FBLN4 in human disease, we investigated two cohorts of patients. Direct sequencing of <em>17</em> patients with cutis laxa revealed no FBLN4 mutations. In a second group of 22 patients presenting with arterial tortuosity, stenosis and aneurysms, FBLN4 mutations were identified in three patients, two homozygous missense mutations (p.Glu126Lys and p.Ala397Thr) and compound heterozygosity for missense mutation p.Glu126Val and frameshift mutation c.577delC. Immunoblotting analysis showed a decreased amount of fibulin-4 protein in the <em>fibroblast</em> culture media of two patients, a finding sustained by diminished fibulin-4 in the extracellular matrix of the aortic wall on immunohistochemistry. pSmad2 and CTGF immunostaining of aortic and lung tissue revealed an increase in transforming <em>growth</em> <em>factor</em> (TGF)beta signaling. This was confirmed by pSmad2 immunoblotting of <em>fibroblast</em> cultures. In conclusion, patients with recessive FBLN4 mutations are predominantly characterized by aortic aneurysms, arterial tortuosity and stenosis. This confirms the important role of fibulin-4 in vascular elastic fiber assembly. Furthermore, we provide the first evidence for the involvement of altered TGFbeta signaling in the pathogenesis of FBLN4 mutations in humans.

OBJECTIVE

Oncogenic fusions consisting of fibroblast growth factor receptor (FGFR) and TACC are present in a subgroup of glioblastoma (GBM) and other human cancers and have been proposed as new therapeutic targets. We analyzed frequency and molecular features of FGFR-TACC fusions and explored the therapeutic efficacy of inhibiting FGFR kinase in GBM and grade II and III glioma.

METHODS

Overall, 795 gliomas (584 GBM, 85 grades II and III with wild-type and 126 with IDH1/2 mutation) were screened for FGFR-TACC breakpoints and associated molecular profile. We also analyzed expression of the FGFR3 and TACC3 components of the fusions. The effects of the specific FGFR inhibitor JNJ-42756493 for FGFR3-TACC3-positive glioma were determined in preclinical experiments. Two patients with advanced FGFR3-TACC3-positive GBM received JNJ-42756493 and were assessed for therapeutic response.

RESULTS

Three of 85 IDH1/2 wild-type (3.5%) but none of 126 IDH1/2-mutant grade II and III gliomas harbored FGFR3-TACC3 fusions. FGFR-TACC rearrangements were present in 17 of 584 GBM (2.9%). FGFR3-TACC3 fusions were associated with strong and homogeneous FGFR3 immunostaining. They are mutually exclusive with IDH1/2 mutations and EGFR amplification, whereas they co-occur with CDK4 amplification. JNJ-42756493 inhibited growth of glioma cells harboring FGFR3-TACC3 in vitro and in vivo. The two patients with FGFR3-TACC3 rearrangements who received JNJ-42756493 manifested clinical improvement with stable disease and minor response, respectively.

CONCLUSIONS

RT-PCR sequencing is a sensitive and specific method to identify FGFR-TACC-positive patients. FGFR3-TACC3 fusions are associated with uniform intratumor expression of the fusion protein. The clinical response observed in the FGFR3-TACC3-positive patients treated with an FGFR inhibitor supports clinical studies of FGFR inhibition in FGFR-TACC-positive patients.

OBJECTIVE

Fibroblast growth factor (FGF)-21 improves insulin sensitivity and lipid metabolism in obese or diabetic animal models, while human studies revealed increased FGF-21 levels in obesity and type 2 diabetes. Given that FGF-21 has been suggested to be a peroxisome proliferator-activator receptor (PPAR) alpha-dependent regulator of fasting metabolism, we hypothesized that free fatty acids (FFAs), natural agonists of PPARalpha, might modify FGF-21 levels.

METHODS

The effect of fatty acids on FGF-21 was investigated in vitro in HepG2 cells. Within a randomized controlled trial, the effects of elevated FFAs were studied in 21 healthy subjects (13 women and 8 men). Within a clinical trial including 17 individuals, the effect of insulin was analyzed using an hyperinsulinemic-euglycemic clamp and the effect of PPARgamma activation was studied subsequently in a rosiglitazone treatment trial over 8 weeks.

RESULTS

Oleate and linoleate increased FGF-21 expression and secretion in a PPARalpha-dependent fashion, as demonstrated by small-interfering RNA-induced PPARalpha knockdown, while palmitate had no effect. In vivo, lipid infusion induced an increase of circulating FGF-21 in humans, and a strong correlation between the change in FGF-21 levels and the change in FFAs was observed. An artificial hyperinsulinemia, which was induced to delineate the potential interaction between elevated FFAs and hyperinsulinemia, revealed that hyperinsulinemia also increased FGF-21 levels in vivo, while rosiglitazone treatment had no effect.

CONCLUSIONS

The results presented here offer a mechanism explaining the induction of the metabolic regulator FGF-21 in the fasting situation but also in type 2 diabetes and obesity.

Cardiac <em>fibroblasts</em> impact myocardial development and remodeling through intercellular contact with cardiomyocytes, but less is known about noncontact, profibrotic signals whereby <em>fibroblasts</em> alter cardiomyocyte behavior. <em>Fibroblasts</em> and cardiomyocytes were harvested from newborn rat ventricles and separated by serial digestion and gradient centrifugation. Cardiomyocytes were cultured in 1) standard medium, 2) standard medium diluted 1:1 with PBS, or 3) standard medium diluted 1:1 with medium conditioned>> or =72 h by cardiac <em>fibroblasts</em>. Serum concentrations were held constant under all media conditions, and complete medium exchanges were performed daily. Cardiomyocytes began contracting within 24 h at clonal or mass densities with <5% of cells expressing vimentin. Immunocytochemical analysis revealed progressive expression of alpha-smooth muscle actin in cardiomyocytes after 24 h in all conditions. Only cardiomyocytes in <em>fibroblast</em>-conditioned medium stopped contracting by 72 h. There was a significant, sustained increase in vimentin expression specific to these cultures (means +/- SD: conditioned 46.3 +/- 6.0 vs. control 5.3 +/- 2.9%, P < 0.00025) typically with cardiac myosin heavy chain coexpression. Proteomics assays revealed 10 cytokines (VEGF, GRO/KC, monocyte chemoattractant protein-1, leptin, macrophage inflammatory protein-1alpha, IL-6, IL-10, IL-12p70, IL-<em>17</em>, and tumor necrosis <em>factor</em>-alpha) at or below detection levels in unconditioned medium that were significantly elevated in <em>fibroblast</em>-conditioned medium. Latent transforming <em>growth</em> <em>factor</em>-beta and RANTES were present in unconditioned medium but rose to higher levels in conditioned medium. Only granulocyte-macrophage colony-stimulating <em>factor</em> was present above threshold levels in standard medium but decreased with <em>fibroblast</em> conditioning. These data indicated that under the influence of <em>fibroblast</em>-conditioned medium, cardiomyocytes exhibited marked hypertrophy, diminished contractile capacity, and phenotype plasticity distinct from the dedifferentiation program present under standard culture conditions.

OBJECTIVE

Glioma cell migration is determined by a complex interplay between soluble motogens and extracellular matrix components. Several growth factors are thought to be involved in glioma cell migration; however, little is known about their motogenic potency relative to one another.

METHODS

Using modified Boyden chamber assays, we compared the chemotactic effects of scatter factor/hepatocyte growth factor (SF/HGF), transforming growth factor (TGF)-alpha, TGF-beta1, TGF-beta2, epidermal growth factor (EGF), fibroblast growth factor (FGF)-1, FGF-2, insulin-like growth factor (IGF)-1, IGF-2, platelet-derived growth factor (PDGF)-AA, PDGF-BB, vascular endothelial growth factor (VEGF), pleiotrophin (PTN), and midkine (MK) in concentrations ranging from 1 pmol/L to 50 nmol/L on three different human glioblastoma cell lines. Checkerboard analyses distinguished between chemotaxis and chemokinesis. We further investigated the motogenic effects on human cerebral microvascular endothelial cells and analyzed receptor expression profiles.

RESULTS

SF/HGF was the most potent chemotactic factor for all three glioblastoma cell lines, inducing up to 33-fold stimulation of migration. TGF-alpha showed the second strongest effect (up to 17-fold stimulation), and FGF-1 was also chemotactic for all three glioblastoma cell lines analyzed (maximal 4-fold effect). EGF, FGF-2, IGF-1, IGF-2, TGF-beta1, and TGF-beta2 were chemotactic for one or two of the cell lines (2- to 4-fold effects), whereas PDGF-AA, PDGF-BB, VEGF, PTN, and MK had no effect. In contrast, the most potent stimulators of cerebral microvascular endothelial cell migration were PDGF-AA (4-fold) and PDGF-BB (6-fold).

CONCLUSIONS

The expression levels of SF/HGF and TGF-alpha as well as their respective receptors, MET and EGFR, are known to correlate with glioma malignancy grade. The particularly strong motogenic effects of these two growth factors suggest that they could be promising targets for an antimigratory component of glioma therapy, at least in comparison with the 12 other factors that were analyzed.

Directed cell migration is essential for a variety of important biological processes ranging from development and angiogenesis to metastasis. Ras plays a pivotal role in the signaling cascade that governs chemotaxis of <em>fibroblasts</em> toward platelet-derived <em>growth</em> <em>factor</em>-BB (PDGF-BB). Ras activates multiple downstream pathways, which include the extracellular signal-regulated kinase (ERK), Rac, and Ral signaling cascades. We therefore investigated the role of the Rac and ERK pathways in cell migration. We showed that migration of <em>fibroblasts</em> toward PDGF-BB is inhibited by expression of dominant negative Asn-<em>17</em> Rac1. Blocking of the ERK pathway by either expression of dominant negative Ala-218/Ala-222-mitogen-activated protein kinase kinase (A218/A222-MEK1) or by a MEK-specific inhibitor did not inhibit migration toward PDGF-BB. In contrast, migration toward soluble fibronectin was suppressed by inhibition of the ERK pathway but not by Asn-<em>17</em> Rac1 expression. These results indicate that directed cell migration mediated by different receptor classes in response to different ligands differentially utilizes the Rac and ERK pathways and suggest that Rac might play a critical role in pathological processes such as angiogenesis and metastasis.

<AbstractText>Pegbelfermin (BMS-986036), a PEGylated human <em>fibroblast</em> <em>growth</em> <em>factor</em> 21 (FGF21) analogue, has previously been shown to improve markers of metabolism and liver fibrosis in obese patients with type 2 diabetes. In this phase 2a study, we aimed to evaluate the safety and efficacy of pegbelfermin in patients with non-alcoholic steatohepatitis.</AbstractText><p><div><b>METHODS</b></div>In this multicentre, randomised, double-blind, placebo-controlled, parallel-group, phase 2a study, we recruited adults (aged 21-75 years) with a body-mass index of at least 25 kg/m², biopsy-confirmed non-alcoholic steatohepatitis (fibrosis stage 1-3), and a hepatic fat fraction of at least 10% when assessed by magnetic resonance imaging-proton density fat fraction. These patients were enrolled at <em>17</em> medical centres in the USA. Eligible patients were stratified by type 2 diabetes status and they were randomly assigned (1:1:1) by a computer-based system to receive subcutaneous injections of placebo once a day, 10 mg pegbelfermin once a day, or 20 mg pegbelfermin once a week, all for 16 weeks. Participants, the study team administering treatment, and investigators analysing outcomes (who were independent of the study team and had no further involvement) were masked to treatment groups. The primary outcomes were safety and the absolute change in hepatic fat fraction after 16 weeks of treatment. All patients who were randomly assigned to groups and received the study drug or placebo were included in the primary analyses. This trial was registered with ClinicalTrials.gov, number NCT02413372.</p><AbstractText>Between May 12, 2015, and Aug 4, 2016, 184 overweight or obese patients with non-alcoholic steatohepatitis were screened for study inclusion. Of these, 95 (52%) patients were excluded because they no longer met study criteria and 80 (43%) patients entered the placebo lead-in phase. After further exclusions, 75 (94%) patients were randomly assigned to groups, received at least one dose of treatment (25 patients to receive 10 mg pegbelfermin once a day; 24 patients to receive 20 mg pegbelfermin once a week, and 26 patients to receive placebo), and were included in the primary analysis. A prespecified interim analysis at week 8 showed a greater than expected change in the primary outcome and supported early closing of patient enrolment, since this analysis indicated that the full planned sample size was not needed. We observed a significant decrease in absolute hepatic fat fraction in the group receiving 10 mg pegbelfermin daily (-6·8% vs -1·3%; p=0·0004) and in the group receiving 20 mg pegbelfermin weekly (-5·2% vs -1·3%; p=0·008) compared with the placebo group. Most adverse events were mild; the most common events were diarrhoea in eight (16%) of 49 patients treated with pegbelfermin and two (8%) of 26 patients treated with placebo and nausea in seven (14%) patients treated with pegbelfermin and two (8%) patients treated with placebo. There were no deaths, discontinuations due to adverse events, or treatment-related serious adverse events.</AbstractText><AbstractText>Treatment with subcutaneously administered pegbelfermin for 16 weeks was generally well tolerated and significantly reduced hepatic fat fraction in patients with non-alcoholic steatohepatitis. Further study of pegbelfermin is warranted in patients with non-alcoholic steatohepatitis. Additional studies that use liver biopsies would allow for the assessment of pegbelfermin's effects on liver histology. Moreover, further studies should allow assessments of the safety and effectiveness of pegbelfermin in a larger number of patients.</AbstractText><AbstractText>Bristol-Myers Squibb.</AbstractText>

The presence of the intermediate filament protein nestin has been the predominant marker used to describe stem and progenitor cells in the mammalian CNS. In this study, a 998-bp fragment in the 3' region of the nestin mRNA was cloned from human fetal brain cells (HFBC). The nucleotide sequence of the cloned cDNA revealed 21 differences with the previously published human nestin sequence, resulting in <em>17</em> amino acid changes. A 150-amino-acid fragment derived from the cloned nestin cDNA was coupled to glutathione S-transferase and used as an immunogen to generate a rabbit polyclonal antiserum that selectively detects human nestin. HFBC that proliferated in response to basic <em>fibroblast</em> <em>growth</em> <em>factor</em> incorporated 5-bromo-2'-deoxyuridine into their nuclei and immunostained for nestin, indicating nestin expression in proliferating CNS progenitor cells. In all cell cultures, nestin costained with the neuroepithelial cell marker vimentin. A small subset of nestin-stained cells (1-2%) immunostained with neuronal marker MAP-2 during the first week and after 4 weeks in culture. However, during the first week in culture, approximately 10-30% of the total cell population of HFBC stained for the glial cell marker GFAP, and nearly all coimmunostained for nestin. After 4 weeks in culture, a subset of GFAP-positive cells emerged that no longer costained with nestin. These results describe nestin expression not only in CNS progenitor cells but also in the cells which were in transition from a progenitor stage to glial differentiation. Collectively, these data suggest a differential temporal regulation of nestin expression during glial and neuronal cell differentiation.

MCF-7 human breast cancer cells release polypeptides related to insulin-like <em>growth</em> <em>factor</em>-I (IGF-I) and epidermal <em>growth</em> <em>factor</em> (EGF) into serum-free culture medium (CM). Treatment of MCF-7 cells with <em>17</em> beta-estradiol, which is required in vivo for MCF-7 tumor <em>growth</em> in the nude mouse and stimulates the MCF-7 <em>growth</em> rate in vitro, resulted in selectively enhanced <em>growth</em> <em>factor</em> activities in CM. Autostimulatory <em>growth</em>-promoting activity was elevated at least 2-fold, and EGF-like polypeptides were elevated 5-fold but IGF-I immunoreactivity was not elevated. Several species of estrogen-induced receptor-reactive EGF-like polypeptides, suggestive of high molecular weight transforming <em>growth</em> <em>factor</em> alpha, were detected after gel exclusion chromatography of CM extracted with 1 M acetic acid. A 30,000 mol wt peak of EGF receptor competing activity comigrated with a peak of autostimulatory and <em>fibroblast</em>-transforming activity. It is possible that estradiol stimulation of MCF-7 <em>growth</em> and/or tumor formation may depend on induction of EGF-related polypeptide <em>growth</em> <em>factors</em>. EGF-I- and EGF-related polypeptides may act together as autocrine or paracrine <em>growth</em> <em>factors</em> in breast cancer.

OBJECTIVE

The primary objective of this study was to determine whether intracoronary administration of the adenoviral gene for fibroblast growth factor (Ad5FGF-4) can improve myocardial perfusion compared with placebo.

BACKGROUND

Animal studies and observational clinical studies have shown improvement in perfusion of the ischemic myocardium using genes encoding angiogenic growth factors; however, randomized, double-blind data in humans are lacking.

METHODS

We performed a randomized, double-blind, placebo-controlled trial of intracoronary injection of 10(10) adenoviral particles containing a gene encoding fibroblast growth factor (Ad5FGF-4) to determine the effect on myocardial perfusion. Fifty-two patients with stable angina and reversible ischemia comprising >9% of the left ventricle on adenosine single-photon emission computed tomography (SPECT) imaging were randomized to gene therapy (n = 35) or placebo (n = 17). Clinical follow-up was performed, and 51 (98%) patients underwent a second adenosine SPECT scan after 8 weeks.

RESULTS

Overall (n = 52), the mean total perfusion defect size at baseline was 32.4% of the left ventricle, with 20% reversible ischemia and 12.5% scar. At eight weeks, Ad5FGF-4 injection resulted in a significant reduction of ischemic defect size (4.2% absolute, 21% relative; p < 0.001) and placebo-treated patients had no improvement (p = 0.32). Although the change in reversible perfusion defect size between Ad5FGF-4 and placebo was not significant (4.2% vs. 1.6%, p = 0.14), when a single outlier was excluded a significant difference was observed (4.2% vs. 0.8%, p < 0.05). Ad5FGF-4 was well tolerated and did not result in any permanent adverse sequelae.

CONCLUSIONS

Intracoronary injection of Ad5FGF-4 showed an encouraging trend for improved myocardial perfusion; however, further studies of therapeutic angiogenesis with Ad5FGF-4 will be necessary.

The binding interactions for the three primary reactants of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) system, basic FGF (bFGF), an FGF receptor, FGFR1, and the co<em>factor</em> heparin/heparan sulfate (HS), were explored by isothermal titrating calorimetry, ultracentrifugation, and molecular modeling. The binding reactions were first dissected into three binary reactions: (1) FGFR1 + bFGF<=>>FGFR1/bFGF, K1 = 41 (+/- 12) nM; (2) FGFR1 + HS<=>>FGFR1/HS, K2 = 104 (+/- <em>17</em>) microM; and (3) bFGF + HS<=>>bFGF/HS, K3 = 470 (+/- 20) nM, where HS = low MW heparin, approximately 3 kDa. The first, binding of bFGF to FGFR1 in the absence of HS, was found to be a simple binary binding reaction that is enthalpy dominated and characterized by a single equilibrium constant, K1. The conditional reactions of bFGF and FGFR1 in the presence of heparin were then examined under conditions that saturate only the bFGF heparin site (1.5 equiv of HS/bFGF) or saturate the HS binding sites of both bFGF and FGFR1 (1.0 mM HS). Both 3-and 5-kDa low MW heparins increased the affinity for FGFR1 binding to bFGF by approximately 10-fold (Kd = 4.9 +/- 2.0 nM), relative to the reaction with no HS. In addition, HS, at a minimum of 1.5 equiv/bFGF, induced a second FGFR1 molecule to bind to another lower affinity secondary site on bFGF (K4 = 1.9 +/- 0.7 microM) in an entropy-dominated reaction to yield a quaternary complex containing two FGFR1, one bFGF, and at least one HS. Molecular weight estimates by analytical ultracentrifugation of such fully bound complexes were consistent with this proposed composition. To understand these binding reactions in terms of structural components of FGFR1, a three-dimensional model of FGFR1 was constructed using segment match modeling. Electrostatic potential calculations confirmed that an elongated cluster, approximately 15 x 35 A, of nine cationic residues focused positive potential (+2kBT) to the solvent-exposed beta-sheet A, B, E, C' surface of the D(II) domain model, strongly implicating this locus as the HS binding region of FGFR1. Structural models for HS binding to FGFR1, and HS binding to bFGF, were built individually and then assembled to juxtapose adjacent binding sites for receptor and HS on bFGF, against matching proposed <em>growth</em> <em>factor</em> and HS binding sites on FGFR1. The calorimetric binding results and the molecular modeling exercises suggest that bFGF and HS participate in a concerted bridge mechanism for the dimerization of FGFR1 in vitro and presumably for mitogenic signal transduction in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of cytokine expression represents a potentially useful approach for the treatment of idiopathic pulmonary fibrosis (IPF). To identify potential targets for such intervention, semi-quantitative reverse transcriptase-polymerase chain reaction was used to compare the expression of messenger ribonucleic acids (mRNAs) coding for <em>17</em> cytokines in lung tissue obtained from patients with IPF at the time of diagnosis and control subjects. Some cytokines were also studied at the protein level by immunohistochemical techniques. mRNAs coding for all of the cytokines evaluated were detected in both control and fibrotic lung samples. Only transforming <em>growth</em> <em>factor</em> (TGF)-beta and interleukin (IL)-10 mRNAs were quantitatively increased in lung biopsies from patients with IPF compared with those of controls, results confirmed at the protein level by immunohistochemistry. Although mRNAs for platelet-derived <em>growth</em> <em>factor</em> (PDGF)-BB and keratinocyte <em>growth</em> <em>factor</em> (KGF) were expressed in similar amounts in lungs from patients with IPF and controls, localised accumulation of both <em>factors</em> was also observed in IPF. Hyperplastic alveolar epithelial cells were a prominent source of cytokines, where IL-10, PDGF-BB and KGF were present in increased amounts, although increased accumulation in <em>fibroblasts</em>, smooth-muscle cells and matrix components was also observed (PDGF-BB, TGF-beta). These results offer new insights into the cytokines produced in the lung in idiopathic pulmonary fibrosis and suggest that modulation of the production of transforming <em>growth</em> <em>factor</em>-beta and interleukin-10 may represent a potentially useful therapeutic strategy for this disabling disease.

BACKGROUND

Anti-angiogenic treatment is believed to have at least cystostatic effects in highly vascularized tumours like pancreatic cancer. In this study, the treatment effects of the angiogenesis inhibitor Cilengitide and gemcitabine were compared with gemcitabine alone in patients with advanced unresectable pancreatic cancer.

METHODS

A multi-national, open-label, controlled, randomized, parallel-group, phase II pilot study was conducted in 20 centers in 7 countries. Cilengitide was administered at 600 mg/m2 twice weekly for 4 weeks per cycle and gemcitabine at 1000 mg/m2 for 3 weeks followed by a week of rest per cycle. The planned treatment period was 6 four-week cycles. The primary endpoint of the study was overall survival and the secondary endpoints were progression-free survival (PFS), response rate, quality of life (QoL), effects on biological markers of disease (CA 19.9) and angiogenesis (vascular endothelial growth factor and basic fibroblast growth factor), and safety. An ancillary study investigated the pharmacokinetics of both drugs in a subset of patients.

RESULTS

Eighty-nine patients were randomized. The median overall survival was 6.7 months for Cilengitide and gemcitabine and 7.7 months for gemcitabine alone. The median PFS times were 3.6 months and 3.8 months, respectively. The overall response rates were 17% and 14%, and the tumor growth control rates were 54% and 56%, respectively. Changes in the levels of CA 19.9 went in line with the clinical course of the disease, but no apparent relationships were seen with the biological markers of angiogenesis. QoL and safety evaluations were comparable between treatment groups. Pharmacokinetic studies showed no influence of gemcitabine on the pharmacokinetic parameters of Cilengitide and vice versa.

CONCLUSIONS

There were no clinically important differences observed regarding efficacy, safety and QoL between the groups. The observations lay in the range of other clinical studies in this setting. The combination regimen was well tolerated with no adverse effects on the safety, tolerability and pharmacokinetics of either agent.

BACKGROUND

In situ cellular reprogramming offers the possibility of regenerating functional cardiomyocytes directly from scar fibroblasts, obviating the challenges of cell implantation. We hypothesized that pretreating scar with gene transfer of the angiogenic vascular endothelial growth factor (VEGF) would enhance the efficacy of this strategy.

RESULTS

Gata4, Mef2c, and Tbx5 (GMT) administration via lentiviral transduction was demonstrated to transdifferentiate rat fibroblasts into (induced) cardiomyocytes in vitro by cardiomyocyte marker studies. Fisher 344 rats underwent coronary ligation and intramyocardial administration of an adenovirus encoding all 3 major isoforms of VEGF (AdVEGF-All6A(+)) or an AdNull control vector (n=12/group). Lentivirus encoding GMT or a GFP control was administered to each animal 3 weeks later, followed by histologic and echocardiographic analyses. GMT administration reduced the extent of fibrosis by half compared with GFP controls (12 ± 2% vs 24 ± 3%, P<0.01) and reduced the number of myofibroblasts detected in the infarct zone by 4-fold. GMT-treated animals also demonstrated greater density of cardiomyocyte-specific marker beta myosin heavy chain 7(+) cells compared with animals receiving GFP with or without VEGF (P<0.01). Ejection fraction was significantly improved after GMT vs GFP administration (12 ± 3% vs -7 ± 3%, P<0.01). Eight (73%) GFP animals but no GMT animals demonstrated decreased ejection fraction during this interval (P<0.01). Also, improvement in ejection fraction was 4-fold greater in GMT/VEGF vs GMT/null animals (17 ± 2% vs 4 ± 1%, P<0.05).

CONCLUSIONS

VEGF administration to infarcted myocardium enhances the efficacy of GMT-mediated cellular reprogramming in improving myocardial function and reducing the extent of myocardial fibrosis compared with the use of GMT or VEGF alone.

Interleukin 6 (IL-6) induces the acute phase response, differentiation of B cells, proliferation of T cells, thymocytes, hematopoietic progenitors, hybridoma and plasmacytoma cells. Monocytes, T cells, <em>fibroblasts</em>, epithelial and endothelial cells secrete IL-6. Since IL-6 responsive cell-types may participate in the pathogenesis of glomerular inflammation, we studied the secretion of IL-6 by rat MCs, using the IL-6 dependent hybridoma cell line B9. The results of our studies indicate that MCs secrete IL-6 with a molecular weight of <em>17</em>-42 kDa and isoelectric point of 4.0 to 5.3 MC-IL-6 activity could be blocked by a polyclonal antimurine-IL-6 antibody. MC express IL-6 mRNA as determined by Northern blot. Furthermore, our data demonstrate that IL-6 acts as an autocrine <em>growth</em> <em>factor</em> for MC. Incubation of subconfluent MC with recombinant IL-6 results in a dose-dependent increase of 3H-thymidine incorporation and number of MCs. Moreover, reverse phase HPLC fractions of MC-CM containing IL-6 activity increase 3H-thymidine incorporation by MC. In addition to its possible paracrine role in mediating the immune response in the glomerulus, MC-IL-6 may also be one of the autocrine signals leading to mesangial cell proliferation in vivo.

The multidrug resistance gene 1 (MDR1) product, P-glycoprotein (P-gp), pumps out a variety of anticancer agents from the cell, including anthracyclines, Vinca alkaloids, and taxanes. The expression of P-gp therefore confers resistance to these anticancer agents. In our present study, we found that FTI-277 (a farnesyltransferase inhibitor), U0126 [an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)], and <em>17</em>-allylamino-<em>17</em>-demethoxygeldanamycin (an inhibitor of heat shock protein 90) reduced the endogenous expression levels of P-gp in the human colorectal cancer cells, HCT-15 and SW620-14. In contrast, inhibitors of phosphatidylinositol 3-OH kinase, mammalian target of rapamycin, p38 mitogen-activated protein kinase, and c-Jun NH(2)-terminal kinase did not affect P-gp expression in these cells. We further found that U0126 down-regulated exogenous P-gp expression in the MDR1-transduced human breast cancer cells, MCF-7/MDR and MDA-MB-231/MDR. However, the MDR1 mRNA levels in these cells were unaffected by this treatment. PD98059 (a MEK inhibitor), ERK small interfering RNA, and p90 ribosomal S6 kinase (RSK) small interfering RNA also suppressed P-gp expression. Conversely, epidermal <em>growth</em> <em>factor</em> and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> enhanced P-gp expression, but the MDR1 mRNA levels were unchanged in epidermal <em>growth</em> <em>factor</em>-stimulated cells. Pulse-chase analysis revealed that U0126 promoted P-gp degradation but did not affect the biosynthesis of this gene product. The pretreatment of cells with U0126 enhanced the paclitaxel-induced cleavage of poly(ADP-ribose) polymerase and paclitaxel sensitivity. Furthermore, U0126-treated cells showed high levels of rhodamine123 uptake. Hence, our present data show that inhibition of the MEK-ERK-RSK pathway down-regulates P-gp expression levels and diminishes the cellular multidrug resistance.

Type I and type III procollagen are reduced in photodamaged human skin. This reduction could result from increased degradation by metalloproteinases and/or from reduced procollagen synthesis. In the present study, we investigated type I procollagen production in photodamaged and sun-protected human skin. Skin samples from severely sun-damaged forearm skin and matched sun-protected hip skin from the same individuals were assessed for type I procollagen gene expression by in situ hybridization and for type I procollagen protein by immunostaining. Both mRNA and protein were reduced ( approximately 65 and 57%, respectively) in photodamaged forearm skin compared to sun-protected hip skin. We next investigated whether reduced type I procollagen production was because of inherently reduced capacity of skin <em>fibroblasts</em> in severely photodamaged forearm skin to synthesize procollagen, or whether contextual influences within photodamaged skin act to down-regulate type I procollagen synthesis. For these studies, <em>fibroblasts</em> from photodamaged skin and matched sun-protected skin were established in culture. Equivalent numbers of <em>fibroblasts</em> were isolated from the two skin sites. <em>Fibroblasts</em> from the two sites had similar <em>growth</em> capacities and produced virtually identical amounts of type I procollagen protein. These findings indicate that the lack of type I procollagen synthesis in sun-damaged skin is not because of irreversible damage to <em>fibroblast</em> collagen-synthetic capacity. It follows, therefore, that <em>factors</em> within the severely photodamaged skin may act in some manner to inhibit procollagen production by cells that are inherently capable of doing so. Interactions between <em>fibroblasts</em> and the collagenous extracellular matrix regulate type I procollagen synthesis. In sun-protected skin, collagen fibrils exist as a highly organized matrix. <em>Fibroblasts</em> are found within the matrix, in close apposition with collagen fibers. In photodamaged skin, collagen fibrils are shortened, thinned, and disorganized. The level of partially degraded collagen is approximately 3.6-fold greater in photodamaged skin than in sun-protected skin, and some <em>fibroblasts</em> are surrounded by debris. To model this situation, skin <em>fibroblasts</em> were cultured in vitro on intact collagen or on collagen that had been partially degraded by exposure to collagenolytic enzymes. Collagen that had been partially degraded by exposure to collagenolytic enzymes from either bacteria or human skin underwent contraction in the presence of dermal <em>fibroblasts</em>, whereas intact collagen did not. <em>Fibroblasts</em> cultured on collagen that had been exposed to either source of collagenolytic enzyme demonstrated reduced proliferative capacity (22 and <em>17</em>% reduction on collagen degraded by bacterial collagenase or human skin collagenase, respectively) and synthesized less type I procollagen (36 and 88% reduction, respectively, on a per cell basis). Taken together, these findings indicate that 1) <em>fibroblasts</em> from photoaged and sun-protected skin are similar in their capacities for <em>growth</em> and type I procollagen production; and 2) the accumulation of partially degraded collagen observed in photodamaged skin may inhibit, by an as yet unidentified mechanism, type I procollagen synthesis.

OBJECTIVE

HIV causes inflammation that can be at least partially corrected by HAART. To determine the qualitative and quantitative nature of cytokine perturbation, we compared cytokine patterns in three HIV clinical groups, including HAART responders (HAART), untreated HIV noncontrollers, and HIV-uninfected (NEG).

METHODS

Multiplex assays were used to measure 32 cytokines in a cross-sectional study of participants in the Women's Interagency HIV Study. Participants from three groups were included: HAART (n = <em>17</em>), noncontrollers (n = 14), and HIV NEG (n = <em>17</em>).

RESULTS

Several cytokines and chemokines showed significant differences between noncontrollers and NEG participants, including elevated interferon gamma-induced 10 (IP-10) and tumor necrosis factor-α (TNF-α) and decreased interleukin-12(p40) [IL-12(p40)], IL-15, and fibroblast growth factor-2 (FGF-2) in noncontroller participants. Biomarker levels among HAART women more closely resembled the NEG, with the exception of TNF-α and FGF-2. Secondary analyses of the combined HAART and noncontroller groups revealed that IP-10 showed a strong, positive correlation with viral load and negative correlation with CD4(+) T-cell counts. The growth factors vascular endothelial growth factor, epidermal growth factor, and FGF-2 all showed a positive correlation with increased CD4(+) T-cell counts.

CONCLUSIONS

Untreated, progressive HIV infection was associated with decreased serum levels of cytokines important in T-cell homeostasis (IL-15) and T-cell phenotype determination (IL-12), and increased levels of innate inflammatory mediators such as IP-10 and TNF-α. HAART was associated with cytokine profiles that more closely resembled those of HIV-uninfected women. The distinctive pattern of cytokine levels in the three study groups may provide insights into HIV pathogenesis, and responses to therapy.

OBJECTIVE

In COPD, the airways are chronically inflamed, and we have now observed fragmentation of the reticular basement membrane (Rbm). This appears to be a hallmark of the process known as epithelial mesenchymal transition (EMT), in which epithelial cells migrate through the Rbm and differentiate into fibroblasts. The aim of this study was to confirm the extent and relevance of Rbm fragmentation in smokers and patients with COPD, and to undertake a preliminary analysis of some classical markers of EMT.

METHODS

Endobronchial biopsies from current smokers (CS; n = 17) and ex-smokers with COPD (ES; n = 15), smokers with normal lung function (NS; n = 16) and never-smoking control subjects (NC; n = 15) were stained for the EMT markers, S100A4, vimentin, epidermal growth factor receptor and matrix metalloproteinase-9.

RESULTS

Compared with NC, there was significant Rbm fragmentation in the CS, ES and NS groups, which was positively associated with smoking history in subjects with COPD. Staining for basal epithelial S100A4, epithelial epidermal growth factor receptor and matrix metalloproteinase-9 in cells within Rbm clefts, and for S100A4 in Rbm cells, was increased in the CS, NS and ES groups compared with the NC group. There was also increased Rbm cell S100A4 staining in the CS group compared with the ES and NS groups. Basal epithelial cell staining for S100A4 was inversely correlated with airflow limitation. Double staining for both S100A4 and vimentin further strengthened the likelihood that these changes represented active EMT.

CONCLUSIONS

This is the first detailed description of fragmentation and cellularity of the Rbm in smokers, which were most marked in subjects with COPD. The data are consistent with active EMT in these subjects.

OBJECTIVE

The fibroblast growth factor receptor (FGFR)-3 fusion genes have been recently demonstrated in a subset of non-small cell lung cancer (NSCLC). To aid in identification and treatment of these patients, we examined the frequency, clinicopathologic characteristics, and treatment outcomes of patients who had NSCLC with or without FGFR fusions.

METHODS

Fourteen known FGFR fusion variants, including FGFR1, FGFR2, and FGFR3, were detected by RT-PCR and verified by direct sequencing in 1,328 patients with NSCLC. All patients were also analyzed for mutations in EGFR, KRAS, HER2, BRAF, ALK, RET, and ROS1. Clinical characteristics, including age, sex, smoking status, stage, subtypes of lung adenocarcinoma, relapse-free survival, and overall survival, were collected.

RESULTS

Of 1,328 tumors screened, two (0.2%) were BAG4-FGFR1 fusion and 15 (1.1%) were FGFR3-TACC3 fusion. Six of 1,016 patients with lung adenocarcinoma were FGFR3-TACC3 fusions and 11 of 312 lung squamous cell carcinoma harbored BAG4-FGFR1 or FGFR3-TACC3 fusions. Compared with the FGFR fusion-negative group, patients with FGFR fusions were more likely to be smokers (94.1%, 16 of 17 patients, P < 0.001), significantly associated with larger tumor (>3 cm; 88.2%, 15 of 17 patients, P < 0.001) and with a tendency to be more poorly differentiated (53.9%, nine of 17 patients, P = 0.095).

CONCLUSIONS

FGFR fusions define a molecular subset of NSCLC with distinct clinical characteristics. FGFR is a druggable target and patients with FGFR fusions may benefit from FGFR-targeted therapy, which needs further clinical investigation.

OBJECTIVE

Angiogenesis is a process stimulated in inflamed synovium of patients with osteoarthritis (OA), and contributes to the progression of the disease. Synovial <em>fibroblasts</em> secrete angiogenic <em>factors</em>, such as vascular endothelial <em>growth</em> <em>factor</em> (VEGF), an up-regulator of angiogenesis, and this ability is increased by interleukin (IL)-1beta. The purpose of this study was to verify whether IL-<em>17</em> contributes and/or synergizes with IL-1beta and tumor necrosis <em>factor</em> (TNF)-alpha in vessel development in articular tissues by stimulating the secretion of proangiogenic <em>factors</em> by synovial <em>fibroblasts</em>.

METHODS

We stimulated in vitro synovial <em>fibroblasts</em> isolated from OA, rheumatoid arthritis (RA) and fractured patients (FP) with IL-<em>17</em> and IL-1beta and from OA patients with IL-<em>17</em>, IL-1beta and TNF-alpha. In the supernatants from the cultures, we assayed the amount of VEGF by immunoassay and other angiogenic <em>factors</em> (keratinocyte <em>growth</em> <em>factor</em>, KGF; hepatocyte <em>growth</em> <em>factor</em>, HGF; heparin-binding endothelial <em>growth</em> <em>factor</em>, HB-EGF; angiopoietin-2, Ang-2; platelet-derived <em>growth</em> <em>factor</em> B, PDGF-BB; thrombopoietin, TPO) by chemiluminescence; semiquantitative RT-PCR was used to state mRNA expression of nonreleased angiogenic <em>factors</em> (Ang-2 and PDGF-BB) and tissue inhibitors of metalloproteinase (TIMP)-1.

RESULTS

IL-<em>17</em>, TNF-alpha and IL-1beta increased VEGF secretion by synovial <em>fibroblasts</em> from OA patients. IL-<em>17</em> and IL-1beta also increased VEGF secretion in RA and FP. Besides, IL-<em>17</em> increased KGF and HGF secretions in OA, RA and FP; in OA and RA, IL-<em>17</em> also increased the HB-EGF secretion and the expression of TIMP-1 as protein and mRNA. In OA patients IL-<em>17</em> had an additive effect on TNF-alpha-stimulated VEGF secretion.

CONCLUSIONS

These results suggest that IL-<em>17</em> is an in vitro stimulator of angiogenic <em>factor</em> release, both by its own action and by cooperating with TNF-alpha.

The interaction of heparan sulfate with protein ligands depends on unique oligosaccharide sequences containing iduronic acid (IdUA), N-sulfated glucosamine residues, and O-sulfated sugars. To study the role of O-sulfation in greater detail, we isolated a Chinese hamster ovary cell mutant defective in 2-O-sulfation of iduronic acid. The mutant, pgsF-<em>17</em>, was identified by a colony blotting assay in which colonies of mutagen-treated cells were replica plated to two disks of polyester cloth. One disk was blotted with 125I-labeled basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) to measure binding to cell surface proteoglycans. The other disk was incubated with 35SO4 to measure proteoglycan biosynthesis. Autoradiography revealed a colony that did not bind 125I-bFGF, but incorporated 35SO4 normally (mutant pgsF-<em>17</em>). Complete deaminative cleavage of heparan sulfate revealed that material from pgsF-<em>17</em> lacked IdUA(2OSO3)-GlcNSO3 and IdUA(2OSO3)-GlcNSO3(6OSO3), but contained a higher proportion of glucuronic acid GlcUA-GlcNSO3(6OSO3) and IdUA-GlcNSO3(6OSO3). Assay of the 2-O-sulfotransferase that acts on IdUA residues showed that mutant <em>17</em> lacked enzyme activity. Interestingly, the alteration resulted in accumulation of GlcNSO3 groups, suggesting that under normal conditions 2-O-sulfation decreases GlcNAc N-deacetylation/N-sulfation, and that the reactions occur simultaneously. The formation of IdUA and 6-O-sulfated glucosaminyl residues appears to be independent of 2-O-sulfation. pgsF-<em>17</em> also lacks 2-O-sulfated GlcUA residues, suggesting that the same enzyme is responsible for 2-O-sulfation of IdUA and GlcUA residues. Mutant <em>17</em> provides a useful tool for studying the regulation of heparan sulfate biosynthesis and the relationship of heparan sulfate fine structure to its biological function.

To grow and metastasize, solid tumours must develop their own blood supply by neo-angiogenesis. Thalidomide inhibits the processing of mRNA encoding peptide molecules including tumour necrosis <em>factor</em>-alpha (TNF-alpha) and the angiogenic <em>factor</em> vascular endothelial <em>growth</em> <em>factor</em> (VEGF). This study investigated the use of continuous low dose Thalidomide in patients with a variety of advanced malignancies. Sixty-six patients (37 women and 29 men; median age, 48 years; range 33-62 years) with advanced measurable cancer (19 ovarian, 18 renal, <em>17</em> melanoma, 12 breast cancer) received Thalidomide 100 mg orally every night until disease progression or unacceptable toxicity was encountered. Three of 18 patients with renal cancer showed partial responses and a further three patients experienced stabilization of their disease for up to 6 months. Although no objective responses were seen in the other tumour types, there were significant improvements in patients' sleeping (P < 0.05) and maintained appetite (P < 0.05). Serum and urine concentrations of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), TNF-alpha and VEGF were measured during treatment and higher levels were associated with progressive disease. Thalidomide was well tolerated: Two patients developed WHO Grade 2 peripheral neuropathy and eight patients developed WHO grade 2 lethargy. No patients developed WHO grade 3 or 4 toxicity. Further studies evaluating the use of Thalidomide at higher doses as a single agent for advanced renal cancer and in combination with biochemotherapy regimens are warranted.

A two-site enzyme-linked immunosorbent assay was used to quantitate levels of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> in the vitreous from 36 patients undergoing vitrectomy for a variety of retinal conditions, including proliferative diabetic retinopathy, macular pucker, and retinal detachment with and without proliferative vitreoretinopathy. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> levels ranged from undetectable to 52 ng/mL. In patients with proliferative diabetic retinopathy, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> levels were greater than or equal to 30 ng/mL in 8 of <em>17</em> specimens. Of the 8 patients with elevated basic <em>fibroblast</em> <em>growth</em> <em>factor</em> levels, 6 had evidence of active proliferative disease (ie, neovascularization of the disc or iris), whereas in the patients who had undetectable levels only 2 of 9 had evidence of neovascularization of disc and none had neovascularization of the iris. In the rhegmatogenous retinal detachment group, 2 of 10 eyes had elevated basic <em>fibroblast</em> <em>growth</em> <em>factor</em> levels, while none in the macular pucker group had elevated levels. Our study documents increased levels of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> in vitreous specimens from patients with proliferative diabetic retinopathy, particularly those with active proliferative retinopathy. The role of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> in the pathogenesis of various retinal disease entities is discussed.