Including Fibroblast Growth Factor Research Articles

Glycosaminoglycans compositional analysis of Urodele axolotl (Ambystoma mexicanum) and Porcine Retina.

Retinal degenerative diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), are major causes of blindness worldwide. Humans cannot regenerate retina, however, axolotl (Ambystoma mexicanum), a laboratory-bred salamander, can regenerate retinal tissue throughout adulthood. Classic signaling pathways, including fibroblast growth factor (FGF), are involved in axolotl regeneration. Glycosaminoglycan (GAG) interaction with FGF is required for signal transduction in this pathway. GAGs are anionic polysaccharides in extracellular matrix (ECM) that have been implicated in limb and lens regeneration of amphibians, however, GAGs have not been investigated in the context of retinal regeneration. GAG composition is characterized native and decellularized axolotl and porcine retina using liquid chromatography mass spectrometry. Pig was used as a mammalian vertebrate model without the ability to regenerate retina. Chondroitin sulfate (CS) was the main retinal GAG, followed by heparan sulfate (HS), hyaluronic acid, and keratan sulfate in both native and decellularized axolotl and porcine retina. Axolotl retina exhibited a distinctive GAG composition pattern in comparison with porcine retina, including a higher content of hyaluronic acid. In CS, higher levels of 4- and 6- O-sulfation were observed in axolotl retina. The HS composition was greater in decellularized tissues in both axolotl and porcine retina by 7.1% and 15.4%, respectively, and different sulfation patterns were detected in axolotl. Our findings suggest a distinctive GAG composition profile of the axolotl retina set foundation for role of GAGs in homeostatic and regenerative conditions of the axolotl retina and may further our understanding of retinal regenerative models.

Similarities and Distinctions in the Effects of Metformin and Carbon Monoxide in Immunometabolism.

Immunometabolism, defined as the interaction of metabolic pathways with the immune system, influences the pathogenesis of metabolic diseases. Metformin and carbon monoxide (CO) are two pharmacological agents known to ameliorate metabolic disorders. There are notable similarities and differences in the reported effects of metformin and CO on immunometabolism. Metformin, an anti-diabetes drug, has positive effects on metabolism and can exert anti-inflammatory and anti-cancer effects via adenosine monophosphate-activated protein kinase (AMPK)-dependent and AMPK-independent mechanisms. CO, an endogenous product of heme oxygenase-1 (HO-1), can exert anti-inflammatory and antioxidant effects at low concentration. CO can confer cytoprotection in metabolic disorders and cancer via selective activation of the protein kinase R-like endoplasmic reticulum (ER) kinase (PERK) pathway. Both metformin and CO can induce mitochondrial stress to produce a mild elevation of mitochondrial ROS (mtROS) by distinct mechanisms. Metformin inhibits complex I of the mitochondrial electron transport chain (ETC), while CO inhibits ETC complex IV. Both metformin and CO can differentially induce several protein factors, including fibroblast growth factor 21 (FGF21) and sestrin2 (SESN2), which maintain metabolic homeostasis; nuclear factor erythroid 2-related factor 2 (Nrf2), a master regulator of the antioxidant response; and REDD1, which exhibits an anticancer effect. However, metformin and CO regulate these effects via different pathways. Metformin stimulates p53- and AMPK-dependent pathways whereas CO can selectively trigger the PERK-dependent signaling pathway. Although further studies are needed to identify the mechanistic differences between metformin and CO, pharmacological application of these agents may represent useful strategies to ameliorate metabolic diseases associated with altered immunometabolism.

Behavior of Stem-Like Cells, Precursors for Tissue Regeneration in Urodela, Under Conditions of Microgravity.

We summarize data from our experiments on stem-like cell-dependent regeneration in amphibians in microgravity. Considering its deleterious effect on many tissues, we asked whether microgravity is compatible with reparative processes, specifically activation and proliferation of source cells. Experiments were conducted using tailed amphibians, which combine profound regenerative capabilities with high robustness, allowing an in vivo study of lens, retina, limb, and tail regeneration in challenging settings of spaceflight. Microgravity promoted stem-like cell proliferation to a varying extent (up to 2-fold), and it seemed to speed up source cell dedifferentiation, as well as sequential differentiation in retina, lens, and limb, leading to formation of bigger and more developed regenerates than in 1g controls. It also promoted proliferation and hypertrophy of Müller glial cells, eliciting a response similar to reactive gliosis. A significant increase in stem-like cell proliferation was mostly beneficial for regeneration and only in rare cases caused moderate tissue growth abnormalities. It is important that microgravity yielded a lasting effect even if applied before operations. We hypothesize on the potential mechanisms of gravity-dependent changes in stem-like cell behavior, including fibroblast growth factor 2 signaling pathway and heat shock proteins, which were affected in our experimental settings. Taken together, our data indicate that microgravity does not disturb the natural regenerative potential of newt stem-like cells, and, depending on the system, even stimulates their dedifferentiation, proliferation, and differentiation. We discuss these data along with publications on mammalian stem cell behavior in vitro and invertebrate regeneration in vivo in microgravity. In vivo data are very scarce and require further research using contemporary methods of cell behavior analysis to elucidate mechanisms of stem cell response to altered gravity. They are relevant for both practical applications, such as managing human reparative responses in spaceflight, and fundamental understanding of stem cell biology.

A functional variant alters binding of activating protein 1 regulating expression of FGF7 gene associated with chronic obstructive pulmonary disease

BackgroundGenome-wide association studies (GWASs) of a large cohort of subjects with chronic obstructive pulmonary disease (COPD) have successfully identified multiple risk genes, including fibroblast growth factor 7 (FGF7). However, the underlying molecular mechanism influencing function of FGF7 and risk of COPD remains further study.MethodsIn this study, we replicated the genetic association of variants near the FGF7 gene in 258 Chinese Han patients with COPD and 311 healthy controls. Additionally, we functionally evaluated a candidate causal variant upstream of the FGF7 gene.ResultsThe most significant association was observed at rs12905203 (P = 5.9 × 10− 3, odd ratio, OR = 1.516) that explains associations of previously reported variants at the FGF7 locus. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) assays showed that the risk allele of the variant was bound to activator protein 1 transcription factors (c-Fos and c-Jun) with a significantly reduced affinity and associated with decreased mRNA expression of FGF7 in fibroblast cells at both resting and PMA/Ionomycin-stimulated conditions. Overexpression of c-Fos and c-Jun proteins or stimulation with PMA/Ionomycin significantly increases mRNA expression of FGF7 in fibroblast cells. Bioinformatic analysis showed that the variant overlaps with multiple genetic regulatory marks, suggesting the regulatory DNA element might function as an enhancer for the FGF7 gene. Luciferase enhancer activity assays demonstrated that the DNA sequences carrying the variant produce enhancer activity while the risk allele of the variant reduces its activity.ConclusionsIn this study, we demonstrated a consistent association of the FGF7 gene with COPD and mechanistically characterized a candidate functional variant upstream of the FGF7 gene. These data highlighted the important role of the risk variant and the FGF7 gene in influencing risk for COPD.

A Review of the Contribution of Mast Cells in Wound Healing: Involved Molecular and Cellular Mechanisms.

Mast cells (MCs), apart from their classic role in allergy, contribute to a number of biologic processes including wound healing. In particular, two aspects of their histologic distribution within the skin have attracted the attention of researchers to study their wound healing role; they represent up to 8% of the total number of cells within the dermis and their cutaneous versions are localized adjacent to the epidermis and the subdermal vasculature and nerves. At the onset of a cutaneous injury, the accumulation of MCs and release of proinflammatory and immunomodulatory mediators have been well documented. The role of MC-derived mediators has been investigated through the stages of wound healing including inflammation, proliferation, and remodeling. They contribute to hemostasis and clot formation by enhancing the expression of factor XIIIa in dermal dendrocytes through release of TNF-α, and contribute to clot stabilization. Keratinocytes, by secreting stem cell factor (SCF), recruit MCs to the site. MCs in return release inflammatory mediators, including predominantly histamine, VEGF, interleukin (IL)-6, and IL-8, that contribute to increase of endothelial permeability and vasodilation, and facilitate migration of inflammatory cells, mainly monocytes and neutrophils to the site of injury. MCs are capable of activating the fibroblasts and keratinocytes, the predominant cells involved in wound healing. MCs stimulate fibroblast proliferation during the proliferative phase via IL-4, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) to produce a new extracellular matrix (ECM). MC-derived mediators including fibroblast growth factor-2, VEGF, platelet-derived growth factor (PDGF), TGF-β, nerve growth factor (NGF), IL-4, and IL-8 contribute to neoangiogenesis, fibrinogenesis, or reepithelialization during the repair process. MC activation inhibition and targeting the MC-derived mediators are potential therapeutic strategies to improve wound healing through reduced inflammatory responses and scar formation.

Brain Peptides for the Treatment of Neuropsychiatric Disorders.

The realization of the importance of growth factors in adult CNS led to several studies investigating their roles in neuropsychiatric disorders. Based on the observations that chronic stress decreases brain-derived neurotrophic factor (BDNF) and antidepressant treatments reverse BDNF to normal levels, "neurotrophic hypothesis of depression" was proposed. Subsequent studies found that several other growth factors, including fibroblast growth factor (FGF), vascular endothelial growth factor, nerve growth factor were also decreased by chronic stress. Growth factors promote stem cell survival, angiogenesis and neurogenesis in addition to having anti-apoptotic and anti-inflammatory effects, all of which make them potential drug candidates as neuroprotective or neurorestorative agents. Indeed, certain peptides have consistently been shown to improve stroke outcome in experimental models of cerebral ischemia. Recent developments in nanotechnology appear promising in overcoming the blood-brain barrier and in delivering sufficient amounts of these large peptides to the brain after systemic administration. In addition to the translational potential resulting from application of nanotechnical approaches for delivering these large peptide growth factors, recent success obtained with small molecule and peptide antagonists of calcitonin gene-related peptide has created renewed enthusiasm to elucidate the role of neuropeptides in migraine headache, one of the most common health problems in the world. In this review, we will first focus on the role of FGF2 in mood disorders as well as in ischemic stroke. We will also introduce the nanomedicines developed to efficiently deliver FGF2 to the brain. In the last section, we will explore roles of the neuropeptides in migraine and its acute and prophylactic treatment.

Multifunctional Chitosan Inverse Opal Particles for Wound Healing.

Wound healing is one of the most important and basic issues faced by the medical community. In this paper, we present biomass-composited inverse opal particles with a series of advanced features for drug delivery and wound healing. The particles were derived by using chitosan biomass to negatively replicate spherical colloid crystal templates. Because of the interconnected porous structures, various forms of active drugs, including fibroblast growth factor could be loaded into the void spaces of the inverse opal particles and encapsulated by temperature-responsive hydrogel. This endowed the composited particles with the capability of intelligent drug release through the relatively high temperature caused by the inflammation reaction at wound sites. Because the structural colors and characteristic reflection peaks of the composited inverse opal particles are blue-shifted during the release process, the drug delivery can be monitored in real time. It was demonstrated that the biomass-composited microcarriers were able to promote angiogenesis, collagen deposition, and granulation-tissue formation as well as reduce inflammation and thus significantly contributed to wound healing. These features point to the potential value of multifunctional biomass inverse opal particles in biomedicine.

Abstract 2870: A specific pan-FGFR inhibitor has antitumor activity against prostate cancer patient derived xenografts, PDX, expressing high FGFR1

Abstract Prostate Cancer (PCa) is one of the most commonly diagnosed malignancies in men. Patients with advanced metastatic PCa have effective treatment options, but none of them are curative. Androgen deprivation is the most effective therapy, but growth of the cancer resumes over time in most cases, and the disease progresses to castration-resistant PCa (CRPC). Bone is the main site of CRPC progression. Acquired (or inherent) resistance mechanisms to second line therapy options for CRPC eventually lead to disease recurrence and, ultimately, death. The underlying mechanisms of PCa progression to first or second line therapy options are diverse and include fibroblast growth factor (FGF) axis activation. Indeed, we previously reported that blockade of FGFRs with dovitinib (TKI258) (Novartis Pharmaceuticals), a receptor tyrosine kinase inhibitor (TKI) with potent activity against FGFR1-3 and vascular endothelial growth factor receptor (VEGFR) has clinical activity in men with CRPC and bone metastases (PMID: 25186177), thus providing direction for therapy development of FGFR blockade in PCa. Because dovitinib was withdrawn from the clinic by Novartis, we seek to identify an alternative agent with activity against FGFR1 as a candidate for therapy development. With that goal, we tested the antitumor activity of a specific pan-FGFR TKI, JNJ-42756493 (JNJ) (Janssen Pharmaceutical Companies of Johnson&Johnson) against PCa patients derived xenografts (PDXs) expressing high (MDA PCa 118b) and low (MDA PCa 183) endogenous levels of FGFR1. Because bone is the primary site of CRPC progression we tested the antitumor activity of JNJ against these PDXs growing in the bone of mice. By assessing tumor volume by MRI, we found that JNJ has antitumor activity against MDA PCa 118b but not MDA PCa 183. Immunohistochemical analysis of FGFR1 expression exhibited reduction of FGFR1 in tumors of the treated group compared with vehicle treated group in MDA PCa 118b samples. Both these evidences suggest that FGFR1 is the main driver of PCa progression in this PDX and that JNJ is a potent agent against PCas with high FGFR1 expression.Due to the important role that FGF axis has in bone biology, we assessed the effect of JNJ in the bones of mice without tumors by micro-CT analysis. Interestingly, we observed a reduction in bone parameters including bone volume/ total volume (BV/TV) and trabecular thickness (Tb.Th) in the treated group compared with the vehicle treated group, suggesting FGF axis blockade reduces bone mass. However, we identified an increase in the bone surrounding the tumors in the MDA PCa 118b tumor-bearing bones of mice treated with JNJ. These results highlight the complex role of FGF axis in the PCa-bone interaction and warrant further studies to identify candidate patients for this therapy and markers of response in men treated with FGFR inhibition. Citation Format: Estefania Labanca, Jun Yang, Peter Shepherd, Xinhai Wan, Justin M. Roberts, Michael W. Starbuck, Nora M. Navone. A specific pan-FGFR inhibitor has antitumor activity against prostate cancer patient derived xenografts, PDX, expressing high FGFR1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2870.

Abstract 11: Antitumor activity of lenvatinib in the renal cell carcinoma cell line RENCA model resistant to a VEGF specific inhibitor

Abstract Anti-vascular endothelial growth factor (VEGF) therapies have been in clinical use to treat patients with multiple types of cancers, especially in renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC). However, benefits of anti-VEGF therapies are still limited because of the intrinsic/acquired resistance to the treatment. Lenvatinib mesilate (lenvatinib) is an oral multiple receptor tyrosine kinase (RTK) inhibitor that suppresses the kinase activities of VEGF receptors (VEGFR1-3), in addition to other proangiogenic and oncogenic pathway-related RTKs including fibroblast growth factor receptors (FGFR1-4), the platelet-derived growth factor receptor (PDGFR) α, KIT, and RET. Lenvatinib has been approved as a monotherapy for the treatment of radioiodine-refractory differentiated thyroid cancer and in combination with everolimus for the treatment of patients with RCC treated with one prior anti-VEGF therapy in the US and EU. In this study, we established an anti-VEGF treatment resistant preclinical mouse tumor model. We also examined whether lenvatinib, which targeted multiple receptor tyrosine kinases, showed anti-tumor activity in this model. To establish model resistant to anti-VEGF treatment, an expression vector encoding VEGFR-Fc protein (VEGF decoy) was introduced into a mouse RCC cell line RENCA. First, we confirmed the RENCAVEGFR-Fc transfectants secreted VEGF decoy, which selectively interacted with recombinant VEGF protein but not with ligands to other RTKs in in vitro. Tumor growth in vivo of RENCAVEGFR-Fc was significantly slower than that of RENCAMock. RENCAMock transfectants took 16 days to reach a mean tumor volume (TV) of 500 mm3 whereas RENCAVEGFR-Fc transfectants required 72 days to reach same TV. Furthermore, 72 days after inoculation of RENCAVEGFR-Fc transfectants, we resected tumor tissues and confirmed the expression of VEGF decoy protein. Tumor microvessel density was decreased in resected RENCAVEGFR-Fc tumor compared to the same size of RENCAMock tumor. From the above results, we characterized RENCAVEGFR-Fc tumor was resistant to VEGF decoy. Finally, anti-tumor activity of lenvatinib was challenged against RENCAMock or RENCAVEGFR-Fc tumors. When tumor volume reached approximately 300mm3, 10 mg/kg of lenvatinib was orally administered once daily for 14 days. Treatment of lenvatinib led to almost complete inhibition of tumor growth in both RENCAMock and RENCAVEGFR-Fc models. In conclusion, we developed a mouse RENCAVEGFR-Fc model resistant to anti-VEGF treatment, and lenvatinib showed anti-tumor activity in the resistant model as with RENCAMock model. Additional preclinical studies will be investigated to elucidate the signaling pathways associated with resistance to anti-VEGF treatment, in which lenvatinib may inhibit such as FGFR1-4, PDGFRα, KIT and RET. Citation Format: Kenji Ichikawa, Saori Watanabe Miyano, Yukinori Minoshima, Junji Matsui. Antitumor activity of lenvatinib in the renal cell carcinoma cell line RENCA model resistant to a VEGF specific inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 11.

Abstract 4027: Fibroblast growth factor 2 (FGF2) is associated with the development of resistance to pemetrexed in lung cancer cells

Abstract Pemetrexed (MTA) is a folate antimetabolite used for treatment of non-small cell lung cancer and plural mesothelioma. Although several molecules such as thymidylate synthase (TS) or ATP-binding cassette transporter 11 (ABCC11) have been reported to be involved in the mechanisms of resistance to MTA, precise mechanisms underlying resistance to MTA remains unknown. To elucidate the mechanisms of resistance to MTA in lung cancer, we established MTA resistant clones in PC9 and H1993 lung adenocarcinoma cell lines (PC9-MTA, H1993-MTA). Then, gene expression profiles were compared between PC9 and PC9-MTA cells by using microarray analyses, and several genes including fibroblast growth factor 2 (FGF2) were identified. Elevated expression of FGF2 in PC9-MTA cells and H1993-MTA cells were confirmed by western blot analyses. Knockdown of FGF2 by siRNA partially restored the sensitivity to MTA both in PC9-MTA and H1993-MTA cells. In addition, anti-FGFR1 inhibitor (PD173074) restored MTA sensitivity particularly in PC9-MTA cells. On the other hand, the expression of TS was increased in PC9-MTA and H1993-MTA cells, and the inhibition of TS with the TS-targeting siRNA restored the sensitivity to MTA. However, with regard to the expression of FGF2 and TS, the inhibition of FGF2 did not modify the expression of TS, and vise versa. Furthermore, a simultaneous inhibition of FGF2 and TS by siRNAs in the MTA-resistant cells restored MTA sensitivity to approximately the level observed for the parental cell lines. With regard to the factors related to the signal transduction pathway, the phosphorylation of Erk and Akt in H1993-MTA cells was decreased compared with those in the parental cells. When H1993-MTA cells were treated with FGF2-targeting siRNA or anti-FGFR1 inhibitor, the increase of phosphorylation of ERK and AKT were induced. These data indicated the possibility that both FGF2 and TS could confer MTA resistance in the lung cancer cell lines in an independent manner, and that modification of signal transduction through FGF2-FGFR1 pathway might be involved in the development of MTA resistance in lung cancer cells. Citation Format: Kentaro Miura, Ken-ichi Ito, Takaaki Oba, Asumi Iesato. Fibroblast growth factor 2 (FGF2) is associated with the development of resistance to pemetrexed in lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4027.

Human Aging and Longevity Are Characterized by High Levels of Mitokines.

Mitochondrial stress elicits the production of stress response molecules indicated as mitokines, including fibroblast growth factor 21 (FGF21), growth differentiation factor 15 (GDF15), and humanin (HN). Many diseases are characterized by progressive mitochondrial dysfunction with alterations of mitokine secretion. It is still controversial whether healthy aging and extreme longevity are accompanied by an altered production of mitokines. We analyzed FGF21, HN, and GDF15 plasma levels in 693 subjects aged from 21 to 113 years, and the association of these mitokines with parameters of health status. FGF21, HN, and GDF15 resulted increased in old age, with the highest levels found in centenarians. These molecules are associated with worsened parameters (such as handgrip strength, insulin sensitivity, triglycerides), particularly in 70-year-old persons, and their levels are inversely correlated with survival in the oldest subjects. Considering the positive biological effect of these molecules, our results can be interpreted in the framework of the hormetic paradigm as an attempt of the cells/tissues to cope with a stress that can have beneficial or detrimental effects depending on its intensity. Finally, persons with Down Syndrome (characterized by accelerated aging) have higher levels of GDF15 and HN with respect to their siblings, suggesting that these molecules, especially GDF15, could be considered markers of biological age.

Msx2 Marks Spatially Restricted Populations of Mesenchymal Precursors

Craniofacial development requires a set of patterning codes that define the identities of postmigratory mesenchymal cells in a region-specific manner, in which locally expressed morphogens, including fibroblast growth factors (FGFs) and bone morphogenetic proteins (BMPs), provide instructive cues. Msx2, a bona fide target of BMP signaling, is a transcription factor regulating Runx2 and osterix (Osx), whose mutations are associated with cranial deformities in humans. Here we show that Msx2 defines osteo-chondro precursor cells in specific regions of the craniofacial mesenchyme at the postmigratory stage, particularly in the mandibular process and the posterior cranial vault. Analysis of Msx2-creER mice revealed that early mesenchymal cells in proximity to the BMP4-expressing mesenchyme were marked upon tamoxifen injection, and their descendants contributed to diverse types of mesenchymal cells in the later stage, such as chondrocytes and perichondrial cells of the transient cartilage, as well as osteoblasts and suture mesenchymal cells. By contrast, Osx-creER marked osteoblast precursors at the later stage, and their descendants continued to become osteoblasts well into the postnatal stage. Therefore, Msx2 marks spatially restricted populations of mesenchymal precursor cells with diverse differentiation potential, suggesting that extrinsic molecular cues can dictate the nature of postmigratory mesenchymal cells in craniofacial development.

Neuropilin 1 Mediates Keratinocyte Growth Factor Signaling in Adipose-Derived Stem Cells: Potential Involvement in Adipogenesis

Adipogenesis is regulated by a complex network of molecules, including fibroblast growth factors. Keratinocyte growth factor (KGF) has been previously reported to promote proliferation on rat preadipocytes, although the expression of its specific receptor, FGFR2-IIIb/KGFR, is not actually detected in mesenchymal cells. Here, we demonstrate that human adipose-derived stem cells (ASCs) show increased expression of KGF during adipogenic differentiation, especially in the early steps. Moreover, KGF is able to induce transient activation of ERK and p38 MAPK pathways in these cells. Furthermore, KGF promotes ASC differentiation and supports the activation of differentiation pathways, such as those of PI3K/Akt and the retinoblastoma protein (Rb). Notably, we observed only a low amount of FGFR2-IIIb in ASCs, which seems not to be responsible for KGF activity. Here, we demonstrate for the first time that Neuropilin 1 (NRP1), a transmembrane glycoprotein expressed in ASCs acting as a coreceptor for some growth factors, is responsible for KGF-dependent pathway activation in these cells. Our study contributes to clarify the molecular bases of human adipogenesis, demonstrating a role of KGF in the early steps of this process, and points out a role of NRP1 as a previously unknown mediator of KGF action in ASCs.

Fibroblast growth factor 2 (FGF2) regulates cytoglobin expression and activation of human hepatic stellate cells via JNK signaling

Cytoglobin (CYGB) belongs to the mammalian globin family and is exclusively expressed in hepatic stellate cells (HSCs) in the liver. In addition to its gas-binding ability, CYGB is relevant to hepatic inflammation, fibrosis, and cancer because of its anti-oxidative properties; however, the regulation of CYGB gene expression remains unknown. Here, we sought to identify factors that induce CYGB expression in HSCs and to clarify the molecular mechanism involved. We used the human HSC cell line HHSteC and primary human HSCs isolated from intact human liver tissues. In HHSteC cells, treatment with a culture supplement solution that included fibroblast growth factor 2 (FGF2) increased CYGB expression with concomitant and time-dependent α-smooth muscle actin (αSMA) down-regulation. We found that FGF2 is a key factor in inducing the alteration in both CYGB and αSMA expression in HHSteCs and primary HSCs and that FGF2 triggered the rapid phosphorylation of both c-Jun N-terminal kinase (JNK) and c-JUN. Both the JNK inhibitor PS600125 and transfection of c-JUN–targeting siRNA abrogated FGF2-mediated CYGB induction, and conversely, c-JUN overexpression induced CYGB and reduced αSMA expression. Chromatin immunoprecipitation analyses revealed that upon FGF2 stimulation, phospho-c-JUN bound to its consensus motif (5′-TGA(C/G)TCA), located −218 to −222 bases from the transcription initiation site in the CYGB promoter. Of note, in bile duct–ligated mice, FGF2 administration ameliorated liver fibrosis and significantly reduced HSC activation. In conclusion, FGF2 triggers CYGB gene expression and deactivation of myofibroblastic human HSCs, indicating that FGF2 has therapeutic potential for managing liver fibrosis.

N-tert-butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe (BOC2) inhibits the angiogenic activity of heparin-binding growth factors

The peptides N-tert-butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe (BOC2) and BOC-Met-Leu-Phe (BOC1) are widely used antagonists of formyl peptide receptors (FPRs), BOC2 acting as an FPR1/FPR2 antagonist whereas BOC1 inhibits FPR1 only. Extensive investigations have been performed by using these FPR antagonists as a tool to assess the role of FPRs in physiological and pathological conditions. Based on previous observations from our laboratory, we assessed the possibility that BOC2 may exert also a direct inhibitory effect on the angiogenic activity of vascular endothelial growth factor-A (VEGF-A). Our data demonstrate that BOC2, but not BOC1, inhibits the angiogenic activity of heparin-binding VEGF-A165 with no effect on the activity of the non-heparin-binding VEGF-A121 isoform. Endothelial cell-based bioassays, surface plasmon resonance analysis, and computer modeling indicate that BOC2 may interact with the heparin-binding domain of VEGF-A165, thus competing for heparin interaction and preventing the binding of VEGF-A165 to tyrosine kinase receptor VEGFR2, its phosphorylation and downstream signaling. In addition, BOC2 inhibits the interaction of a variety of heparin-binding angiogenic growth factors with heparin, including fibroblast growth factor 2 (FGF2) whose angiogenic activity is blocked by the compound. Accordingly, BOC2 suppresses the angiogenic potential of human tumor cell lines that co-express VEGF-A and FGF2. Thus, BOC2 appears to act as a novel multi-heparin-binding growth factor antagonist. These findings caution about the interpretation of FPR-focusing experimental data obtained with this compound and set the basis for the design of novel BOC2-derived, FPR independent multi-target angiogenesis inhibitors.

Systemic and Adjuvant Therapies for Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma represents the second most common primary liver cancer and is increasing in incidence. Most patients are diagnosed at an advanced, nonsurgical stage and only about 1 in 5 cases are surgically resectable. Despite surgery, the 5-year survival is low at only 30%. Multifocal, node- or margin-positive disease is at a higher risk of recurrence after resection. There is no level 1 evidence in support of postoperative adjuvant therapy. A recent adjuvant therapy phase III trial from the Partenariat de Recherche en Oncologie Digestive-Actions Concertées dans les Cancers Colo-Rectaux et Digestifs (PRODIGE) group reported no survival advantage with adjuvant gemcitabine and oxaliplatin therapy. Locally advanced or metastatic cholangiocarcinoma is treated with gemcitabine-based systemic chemotherapy with suboptimal response and survival. Integration of local therapy such as focal radiation along with induction chemotherapy is now being investigated in multicenter clinical trials. Recent molecular profiling studies have indicated that about 30% to 40% of intrahepatic cholangiocarcinoma cases have actionable mutations. These include fibroblast growth factor receptor (FGFR), isocitrate dehyrogenase 1 (IDH1), epidermal growth factor receptor (EGFR), and BRAF genetic aberrations. Clinical trials targeting these mutations as well as immune therapy using programmed cell death 1 (PD1) inhibitors indicated a promising early signal showing clinical efficacy.

* Glioblastoma Exosomes for Therapeutic Angiogenesis in Peripheral Ischemia.

Peripheral ischemia as a result of occlusive vascular disease is a widespread problem in patients older than the age of 65. Angiogenic therapies that can induce microvascular growth have great potential for providing a long-lasting solution for patients with ischemia and would provide an appealing alternative to surgical and percutaneous interventions. However, many angiogenic therapies have seen poor efficacy in clinical trials, suggesting that patients with long-term peripheral ischemia have considerable therapeutic resistance to angiogenic stimuli. Glioblastoma is one of the most angiogenic tumor types, inducing robust vessel growth in the area surrounding the tumor. One major angiogenic mechanism used by the tumor cells to induce blood vessel growth is the production of exosomes and other extracellular vesicles that can carry pro-angiogenic and immunomodulatory signals. Here, we explored whether the pro-angiogenic aspects of glioblastoma-derived exosomes could be harnessed to promote angiogenesis and healing in the context of peripheral ischemic disease. We demonstrate that the exosomes derived from glioblastoma markedly enhance endothelial cell proliferation and increase endothelial tubule formation in vitro. An analysis of the microRNA expression using next generation sequencing identified that exosomes contained a high concentration of miR-221. In addition, we found that glioblastoma exosomes contained significant amounts of the proteoglycans glypican-1 and syndecan-4, which can serve as co-receptors for angiogenic factors, including fibroblast growth factor-2 (FGF-2). In a hindlimb ischemia model in mice, we found that the exosomes promoted enhanced revascularization in comparison to control alginate gels and FGF-2 treatment alone. Taken together, our results support the fact that glioblastoma-derived exosomes have powerful effects in increasing revascularization in the context of peripheral ischemia.

Heparan Sulfate Microarray Reveals That Heparan Sulfate-Protein Binding Exhibits Different Ligand Requirements.

Heparan sulfates (HS) are linear sulfated polysaccharides that modulate a wide range of physiological and disease-processes. Variations in HS epimerization and sulfation provide enormous structural diversity, which is believed to underpin protein binding and regulatory properties. The ligand requirements of HS-binding proteins have, however, been defined in only a few cases. We describe here a synthetic methodology that can rapidly provide a library of well-defined HS oligosaccharides. It is based on the use of modular disaccharides to assemble several selectively protected tetrasaccharides that were subjected to selective chemical modifications such as regioselective O- and N-sulfation and selective de-sulfation. A number of the resulting compounds were subjected to enzymatic modifications by 3-O-sulfotransferases-1 (3-OST1) to provide 3-O-sulfated derivatives. The various approaches for diversification allowed one tetrasaccharide to be converted into 12 differently sulfated derivatives. By employing tetrasaccharides with different backbone compositions, a library of 47 HS-oligosaccharides was prepared and the resulting compounds were used to construct a HS microarray. The ligand requirements of a number of HS-binding proteins including fibroblast growth factor 2 (FGF-2), and the chemokines CCL2, CCL5, CCL7, CCL13, CXCL8, and CXCL10 were examined using the array. Although all proteins recognized multiple compounds, they exhibited clear differences in structure-binding characteristics. The HS microarray data guided the selection of compounds that could interfere in biological processes such as cell proliferation. Although the library does not cover the entire chemical space of HS-tetrasaccharides, the binding data support a notion that changes in cell surface HS composition can modulate protein function.

Correction: Physiological characterization of a novel PPAR pan agonist, 2-(4-(5,6-methylenedioxybenzo[d]thiazol-2-yl)-2-methylphenoxy)-2-methylpropanoic acid (MHY2013).

// Hye Jin An 1, 2 , Bonggi Lee 1, 2, 5 , Dae Hyun Kim 1, 2 , Eun Kyeong Lee 1, 2 , Ki Wung Chung 1, 2 , Min Hi Park 1, 2 , Hyoung Oh Jeong 1, 2 , Sung Min Kim 1, 2 , Kyoung Mi Moon 1, 2 , Ye Ra Kim 1, 2 , Seong Jin Kim 1, 2 , Hwi Young Yun 1, 2 , Pusoon Chun 3 , Byung Pal Yu 4 , Hyung Ryong Moon 1, 2 , Hae Young Chung 1, 2 1 College of Pharmacy, Pusan National University, Busan 46241, Republic of Korea 2 Molecular Inflammation Research Center for Aging Intervention (MRCA), Pusan National University, Busan 46241, Republic of Korea 3 College of Pharmacy, Inje University, Gyeongsangnam-do 50834, Republic of Korea 4 Department of Physiology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, USA 5 Korean Medicine (KM)-Application Center, Korea Institute of Oriental Medicine (KIOM), Daegu 41062, Republic of Korea Correspondence to: Hae Young Chung, email: hyjung@pusan.ac.kr Hyung Ryong Moon, email: mhr108@pusan.ac.kr Keywords: MHY2013, PPAR pan agonist, metabolic syndrome, FGF21, adiponectin Received: July 29, 2016 Accepted: December 27, 2016 Published: January 25, 2017 ABSTRACT Recently, agonists targeting multiple peroxisome proliferator-activated receptors (PPARs) have been developed to improve metabolic disorders and minimize the side effects of selective PPAR agonists such as weight gain and dyslipidemia. We newly synthesized six 2-methyl-2-( o -tolyloxy)propanoic acid derivatives based on the structure of a well-known PPAR pan agonist, bezafibrate. Of six compounds, MHY2013 was screened as the strongest activator of three PPAR subtypes based on protein docking simulation and luciferase assays. When treated orally in db/db mice, MHY2013 ameliorated obesity-induced insulin resistance, dyslipidemia, and hepatic steatosis without changes of the body weight and levels of liver and kidney injury markers. MHY2013 decreased the serum triglyceride and fatty acid levels, which is associated with an increase in fatty acid oxidation signaling in the liver and thermogenic signaling on white adipose tissue, respectively. Furthermore, MHY2013 markedly increased serum levels of insulin-sensitizing hormones including fibroblast growth factor 21 (FGF21) and adiponectin. In conclusion, this study suggests that, MHY2013 is a novel PPAR pan agonist that improves obesity-induced insulin resistance, dyslipidemia and hepatic steatosis and elevates insulin-sensitizing hormones in the blood.

Abstract 95: Targeting BRD4 overcomes cetuximab resistance in HNSCC

Abstract Background: Nearly 600,000 people are diagnosed with head and neck cancer worldwide and 60% succumb to the disease within 5 years. The epidermal growth factor receptor (EGFR) is a major driver of HNSCC and in 2006 the EGFR monoclonal antibody cetuximab was FDA-approved for HNSCC treatment. However, cetuximab has not conferred significant long-term benefit compared to chemoradiation in patients with poorly differentiated recurrent-metastatic HNSCC. We and others have demonstrated that cetuximab treatment activates alternative receptor tyrosine kinases (RTKs) including Fibroblast Growth Factor (FGF) receptors, Met and Axl in HNSCC. To circumvent administration of multiple RTK inhibitors in combination with cetuximab, we sought to identify a common molecular target that regulates the expression of these RTKs. Bromodomain-containing protein-4 (BRD4) has been shown to regulate the transcription of RTKs in breast cancer models. Hypothesis: We hypothesized that targeting BRD4 will overcome cetuximab resistance by depleting the expression of alternative RTKs in HNSCC. We further hypothesize that genetic and pharmacological targeting of BRD4 will synergize with cetuximab. Results: Using a phospho-RTK array, 72-hour cetuximab treatment increased or sustained phosphorylated levels of EGFR, HER2, HER3, MET, and AXL in several HNSCC cell lines. Treatment with the BRD4 inhibitor JQ1 abrogated both phosphorylated and total RTK abundance in the presence of cetuximab. Cetuximab and JQ1 robustly decreased phosphorylated Src and induced the senescence marker p21. Phenotypically, cetuximab and JQ1 significantly decreased survival and increased apoptosis in 6 HNSCC cell line models, while the normal oral keratinocyte cell line NOKsi had approximately 10-fold higher IC50s for the BRD4 inhibitors, JQ1 and I-BET762, relative to HNSCC cell lines. Importantly, two HNSCC models of acquired cetuximab resistance exhibited robust sensitivity to pharmacological (JQ1, IBET-762) and genetic (RNAi) BRD4 targeting strategies. Moreover, exogneous overexpression of different RTKs (HER3, ALK, and ROR1) resulted in cetuximab resistance that was reversed upon BRD4 targeting (RNAi and JQ1). Combination of cetuximab and JQ1 in co-culture experiments with T cells decreased the CD4+/CD25+ Treg population and PD-L1 expression on HNSCC cell lines. Further, preliminary findings indicate that JQ1 treatment prevents outgrowth of cetuximab-treated HNSCC patient-derived xenograft models. Conclusion: Our findings indicate that targeting BRD4 decreases the activation and expression of multiple RTKs that mediate resistance to the FDA-approved EGFR inhibitor cetuximab. Furthermore, BRD4 abrogates expression of immunosuppressive markers, making it a promising tumor intrinsic and extrinsic therapeutic strategy for HNSCC. Citation Format: Toni Brand, Yan Zeng, Brandon Leonard, Rachel O' Keefe, Hua Li, Daniel Johnson, Jennifer Grandis, Neil E. Bhola. Targeting BRD4 overcomes cetuximab resistance in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 95. doi:10.1158/1538-7445.AM2017-95