Heparin-binding Growth Factors Research Articles

VEGF – Supplemented extracellular matrix is sufficient to induce endothelial differentiation of human iPSC

Extracellular matrix (ECM) provides a scaffold for cells and tissues, but also supports organogenesis and tissue remodeling. The required instructive properties of the ECM to interact with cells depend on matrix architecture, structural proteins and functional matrix components such as growth factors, providing spatial, chemical and functional cues. Decellularized ECM (dECM) has been proposed as an instructive material that promotes tissue regeneration. We investigated the instructive ECM elements preserved in dECM and necessary to promote endothelial differentiation of human induced pluripotent stem cells (hiPSC). We show that detergent-decellularized human kidney ECM remains structurally intact and carries a number of heparin-binding growth factors, including FGF2, VEGF, BMP2, HGF, EGF, PDGF-BB and TGFβ, albeit at reduced levels compared to native tissues. Clearance of these heparin-binding factors, or heparan-sulfate proteoclycans from ECM resulted in massively reduced differentiation of hiPSC, suggesting that remaining structural dECM proteins such as laminin, collagen or fibronectin alone are not instructive. In contrast, replenishing dECM with VEGF replaced medium-supplemented VEGF and resulted in more efficient differentiation of hiPSC into endothelial cells, and even in the absence of other culture-supplemented differentiation factors dECM alone was superior to geltrex. In conclusion, conditioning of dECM with specific growth factors acting as functional cues may allow to generate functional niches by selective promotion of cell attachment, survival and differentiation.

Expression of the heparin-binding growth factors Midkine and pleiotrophin during ocular development

Midkine (MDK) and Pleiotrophin (PTN) belong to a group of heparin-binding growth factors that has been shown to have pleiotropic functions in various biological processes during development and disease. Development of the vertebrate eye is a multistep process that involves coordinated interactions between neuronal and non-neuronal cells, but very little is known about the potential function of MDK and PTN in these processes. In this study, we demonstrate by section in situ hybridization, the spatiotemporal expression of MDK and PTN during ocular development in chick and mouse. We show that MDK and PTN are expressed in dynamic patterns that overlap in a few non-neuronal tissues in the anterior eye and in neuronal cell layers of the posterior eye. We show that the expression patterns of MDK and PTN are only conserved in a few tissues in chick and mouse but they overlap with the expression of some of their receptors LRP1, RPTPZ, ALK, NOTCH2, ITGβ1, SDC1, and SDC3. The dynamic expression patterns of MDK, PTN and their receptors suggest that they function together during the multistep process of ocular development and they may play important roles in cell proliferation, adhesion, and migration of neuronal and non-neuronal cells.

Glycosaminoglycans from Alzheimer's disease hippocampus have altered capacities to bind and regulate growth factors activities and to bind tau.

Glycosaminoglycans (GAGs), including heparan sulfates and chondroitin sulfates, are major components of the extracellular matrix. Upon interacting with heparin binding growth factors (HBGF), GAGs participate to the maintaintenance of tissue homeostasis and contribute to self-healing. Although several processes regulated by HBGF are altered in Alzheimer’s disease, it is unknown whether the brain GAG capacities to bind and regulate the function of HBGF or of other heparin binding proteins, as tau, are modified in this disease. Here, we show that total sulfated GAGs from hippocampus of Alzheimer’s disease have altered capacities to bind and potentiate the activities of growth factors including FGF-2, VEGF, and BDNF while their capacity to bind to tau is remarkable increased. Alterations of GAG structures and capacities to interact with and regulate the activity of heparin binding proteins might contribute to impaired tissue homeostasis in the Alzheimer’s disease brain.

Spatial Micropatterning of Growth Factors in 3D Hydrogels for Location-Specific Regulation of Cellular Behaviors.

Growth factors are potent stimuli for regulating cell function in tissue engineering strategies, but spatially patterning their presentation in 3D in a facile manner using a single material is challenging. Micropatterning is an attractive tool to modulate the cellular microenvironment with various biochemical and physical cues and study their effects on stem cell behaviors. Implementing heparin's ability to immobilize growth factors, dual-crosslinkable alginate hydrogels are micropatterned in 3D with photocrosslinkable heparin substrates with various geometries and micropattern sizes, and their capability to establish 3D micropatterns of growth factors within the hydrogels is confirmed. This 3D micropatterning method could be applied to various heparin binding growth factors, such as fibroblast growth factor-2, vascular endothelial growth factor, transforming growth factor-betas and bone morphogenetic proteins while retaining the hydrogel's natural degradability and cytocompability. Stem cells encapsulated within these micropatterned hydrogels have exhibited spatially localized growth and differentiation responses corresponding to various growth factor patterns, demonstrating the versatility of the approach in controlling stem cell behavior for tissue engineering and regenerative medicine applications.

Fluorous-tag assisted synthesis of a glycosaminoglycan mimetic tetrasaccharide as a high-affinity FGF-2 and midkine ligand

Here, we present the preparation of a sulfated, fully protected tetrasaccharide derivative following the glycosaminoglycan (GAG)-related sequence GlcNAc-β(1 → 4)-Glc-β(1 → 3). The tetramer was efficiently assembled via an iterative glycosylation strategy using monosaccharide building blocks. A fluorous tag was attached at position 6 of the reducing end unit enabling the purification of reaction intermediates by simple fluorous solid phase extraction. Fluorescence polarization competition experiments revealed that the synthesized tetrasaccharide strongly interacts with two heparin-binding growth factors, midkine and FGF-2 (IC50 of 270 nM and 2.4 µM, respectively). Our data indicate that this type of oligosaccharide derivatives, displaying sulfates, hydrophobic protecting groups and a fluorinated tail can be considered as interesting GAG mimetics for the regulation of relevant carbohydrate-protein interactions.

Exostosin-like 2 regulates FGF2 signaling by controlling the endocytosis of FGF2

BackgroundHeparan sulfate proteoglycans are ubiquitously expressed on cell surfaces and in extracellular matrices, and are engaged in heparin-binding growth factor-related signal transduction. Thus, changes in the amounts, structures, and chain lengths of heparan sulfate have profound effects on aspects of cell growth controlled by heparin-binding growth factors such as FGF2. Exostosin glycosyltransferases (EXT1, EXT2, EXTL1, EXTL2, and EXTL3) control heparan sulfate biosynthesis, and the expression levels of their genes regulate the amounts, chain lengths, and sulfation patterns of heparan sulfate. Unlike EXT1, EXT2, and EXTL3, EXTL2 functions chain termination of heparan sulfate. Here, we examined the importance of EXTL2 in FGF2-dependent signaling. MethodsWe investigated heparan sulfate biosynthesis and FGF2 signaling using four cell lines, EXT1-deficient cells, EXT2-, EXTL2-, or EXTL3-knockdown cells, by HPLC, qRT-PCR, flow cytometry, and western blotting. ResultsReduced expression of either EXT1, EXT2, or EXTL3 decreased heparan sulfate biosynthesis, and consequently suppressed the FGF2-dependent proliferation of mouse L fibroblasts. In contrast, although knockdown of EXTL2 increased the amounts of heparan sulfate, FGF2-dependent proliferation was significantly inhibited because the increased heparan sulfate enhanced the incorporation of FGF2 into the cells. ConclusionsEXTL2 controls FGF2 signaling through regulation of heparan sulfate biosynthesis in a manner distinct from that of other exostosins. General significanceThis study provides new insights into the regulatory mechanisms of FGF2 signaling by EXTL2.

Pilot Screening Study of the Angiogenesis Factor Pleiotrophin (PTN) in Serum Samples from Patients of Various Disease Groups

Introduction: The secreted growth factor pleiotrophin (PTN) belongs to the midkine family of heparin-binding growth factors and is tightly regulated during embryogenesis. In contrast to its very limited expression in normal adult tissues, PTN protein levels are markedly increased in different tumors, with PTN showing mitogenic, chemotactic, transforming, pro-angiogenic, pro-invasive and pro-metastatic activity. However, little is known about PTN upregulation in diseases other than cancer. The aim of this study was to investigate PTN serum levels in patients with various non-malignant chronic or acute disorders, and in pregant women, compared to healthy nonpregnant blood donors as negative control group.Materials and Methods: PTN serum levels were determined by a sandwich ELISA.Results: PTN serum levels were found to be significantly elevated with a p-value of <0.05 in patients with the acute non-malignant disorders, acute inflammation, acute vascular disease and acute trauma. In patients with chronic leukemia and solid tumors, and pregnancy, increased PTN serum levels were detected as well.Conclusion: Beyond its described functions in solid tumors and in the central nervous system, these data indicate that PTN is an acute phase protein in the adult organism and that, due to its upregulation under various non-cancer conditions, special caution must be taken when exploring PTN serum levels as potential tumor marker.

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.

P.3.d.053 - Antipsychotic-induced hyperprolactinemia in a sample of psychotic patients

Currently, there is no widely accepted technique to efficiently and reproducibly grow stem and progenitor cells in vitro. Stem cells require contact with extracellular matrices as well as signals from growth factors to proliferate and to retain their stemness. We have shown a novel tissue culture platform (StemTrix cultureware) that transforms standard tissue culture plasticware into a multi-functional chitosan-based scaffold that supports the expansion of neural stem cells. The StemTrix scaffold is comprised of chitosan with immobilized heparin which in turn tethers heparin-binding growth factors. The scaffold is also coated with an adhesive ECM protein. Here we demonstrate that fibronectin or the RGD peptide contained in fibronectin are equally effective in promoting the adhesion, viability and growth of rat and human neural stem cells. When FGF-2 and heparin-binding EGF are tethered to the StemTrix cultureware neural stem cells grow ∼3 times faster and remain in a more primitive state as determined by both Western Blot and gene expression analyses. Another important feature of this new culture platform is that the NSCs remain in a primitive and proliferative state for 4 days without refreshing the culture medium or providing new growth factors, which represents a 20-fold extension of FGF-2’s biological activity vs when it is freely soluble in the medium. To test the utility of this scaffold for propagating other types of stem cells and progenitors we tethered platelet-derived growth factor (PDGF) and FGF-2 alone and in combination to the scaffold and tested the efficacy of this platform to maintain primary oligodendrocyte progenitors or the CG-4 cell line in a primitive state. Oligodendrocyte progenitors plated onto this multifunctional film proliferated for at least 3 days without providing soluble growth factors while inhibiting the expression of the differentiation marker myelin-basic protein. Oligodendrocyte progenitors proliferated 3 times more rapidly than cells maintained on fibronectin-coated culture substrates in culture medium supplemented with soluble FGF-2 and PDGF. Finally, we show that StemTrix cultureware can be produced using clinical grade components, providing users with a fully defined platform suitable for clinical use that maintains stem cells or progenitors in a more uniform and primitive state.

Src-mediated ligand release-independent EGFR transactivation involves TGF-β–induced Smad3 activation in mesangial cells

A great deal of evidence highlighted the pathophysiologic importance of TGF-β1/Smad3 pathway in masangial extracellular matrix (ECM) accumulation, but some alternative signaling pathways are also involved. TGF-β was shown recently to induce rapid and transient epidermal-like growth factor receptor (EGFR) transactivation and subsequent fibronectin expression via heparin-binding epidermal-like growth factors (HB-EGF) release and binding in mesangial cells, which is independent of Smad2 activation. However, whether TGF-β could induce persistent EGFR transactivation remains to be identified. The present study demonstrates that in addition to transient EGFR transactivation, TGF-β1 can also induce continuous EGFR transactivation by a non-ligand–dependent pathway in rat mesangial cells. This sustained EGFR transactivation is mainly due to Src kinase-mediated persistent EGFR tyrosine phosphorylation at Y845 rather than Y1173. TGF-β1–induced early Smad3 phosphorylation is independent of transient EGFR transactivation and ERK1/2 activation initiated by HB-EGF release, whereas Src–mediated chronic EGFR transactivation and ERK1/2 activation participate in Smad3 activation in a relatively modest and delayed manner. Therefore, the present study further clarifies the mechanisms of EGFR transactivation in the TGF-β–initiated ECM upregulation and raises the possibility that targeting EGFR may provide a viable alternative strategy for inhibiting TGF-β in chronic kidney disease.

Midkine and pleiotrophin concentrations in needle biopsies of breast and lung masses.

Midkine (MDK) and pleiotrophin (PTN) are two closely related heparin-binding growth factors which are overexpressed in a wide variety of human cancers. We hypothesized that the concentrations of these factors in washout of biopsy needles would be higher in breast and lung cancer than in benign lesions. Seventy subjects underwent pre-operative core needle biopsies of 78 breast masses (16 malignancies). In 11 subjects, fine needle aspiration was performed ex vivo on 7 non-small cell lung cancers and 11 normal lung specimens within surgically excised lung tissue. The biopsy needle was washed with buffer for immunoassay. The MDK/DNA and the PTN/DNA ratio in most of the malignant breast masses were similar to the ratios in benign masses except one lobular carcinoma in situ (24-fold higher PTN/DNA ratio than the average benign mass). The MDK/DNA and PTN/DNA ratio were similar in most malignant and normal lung tissue except one squamous cell carcinoma (38-fold higher MDK/DNA ratio than the average of normal lung tissue). Both MDK and PTN are readily measurable in washout of needle biopsy samples from breast and lung masses and levels are highly elevated only in a specific subset of these malignancies.

Tethered growth factors on biocompatible scaffolds improve stemness of cultured rat and human neural stem cells and growth of oligodendrocyte progenitors.

Currently, there is no widely accepted technique to efficiently and reproducibly grow stem and progenitor cells in vitro. Stem cells require contact with extracellular matrices as well as signals from growth factors to proliferate and to retain their stemness. We have shown a novel tissue culture platform (StemTrix cultureware) that transforms standard tissue culture plasticware into a multi-functional chitosan-based scaffold that supports the expansion of neural stem cells. The StemTrix scaffold is comprised of chitosan with immobilized heparin which in turn tethers heparin-binding growth factors. The scaffold is also coated with an adhesive ECM protein. Here we demonstrate that fibronectin or the RGD peptide contained in fibronectin are equally effective in promoting the adhesion, viability and growth of rat and human neural stem cells. When FGF-2 and heparin-binding EGF are tethered to the StemTrix cultureware neural stem cells grow ∼3 times faster and remain in a more primitive state as determined by both Western Blot and gene expression analyses. Another important feature of this new culture platform is that the NSCs remain in a primitive and proliferative state for 4days without refreshing the culture medium or providing new growth factors, which represents a 20-fold extension of FGF-2's biological activity vs when it is freely soluble in the medium. To test the utility of this scaffold for propagating other types of stem cells and progenitors we tethered platelet-derived growth factor (PDGF) and FGF-2 alone and in combination to the scaffold and tested the efficacy of this platform to maintain primary oligodendrocyte progenitors or the CG-4 cell line in a primitive state. Oligodendrocyte progenitors plated onto this multifunctional film proliferated for at least 3days without providing soluble growth factors while inhibiting the expression of the differentiation marker myelin-basic protein. Oligodendrocyte progenitors proliferated 3 times more rapidly than cells maintained on fibronectin-coated culture substrates in culture medium supplemented with soluble FGF-2 and PDGF. Finally, we show that StemTrix cultureware can be produced using clinical grade components, providing users with a fully defined platform suitable for clinical use that maintains stem cells or progenitors in a more uniform and primitive state.

Research progress in osteogenesis and osteogenic mechanism of heparan sulfate

To discuss the role of heparan sulfate (HS) in bone formation and bone remodeling and summarize the research progress in the osteogenic mechanism of HS. The domestic and abroad related literature about HS acting on osteoblast cell line in vitro, HS and HS composite scaffold materials acting on the ani-mal bone defect models, and the effect of HS proteoglycans on bone development were summarized and analyzed. Many growth factors involved in fracture healing especially heparin-binding growth factors, such as fibroblast growth factors, bone morphogenetic protein, and transforming growth factor β, are connected noncovalently with long HS chains. HS proteoglycans protect these proteins from protease degradation and are directly involved in the regulation of growth factors signaling and bone cell function. HS can promote the differentiation of stem cells into osteoblasts and enhance the differentiation of osteoblasts. In bone matrix, HS plays a significant role in promoting the formation, maintaining the stability, and accelerating the mineralization. The osteogenesis of HS is pronounced. HS is likely to become the clinical treatment measures of fracture nonunion or delayed union, and is expected to provide more choices for bone tissue engineering with identification of its long-term safety.

Abstract 4626: Macrophages alter heparan sulfate-modifying enzyme levels, growth factor activity and cancer cell growth in 3D hydrogels

Abstract Tumor progression in both primary and metastatic niches often involves the activation of associated or “reactive” stroma similar to that seen in a wound healing response. The stromal participants include fibroblasts, endothelial and immune cells, including macrophages. Cancer-associated fibroblasts (CAFs) and tumor-associated macrophages communicate with local cells including cancer cells and the surrounding stroma to induce epithelial-to-mesenchymal transition, immunosuppression and increased production of extracellular matrix (ECM) components, growth factors, and ECM-remodeling enzymes. Heparan sulfate (HS)-modifying enzymes produced by tumor-associated stroma play key roles in the delivery of heparin-binding growth factors (HBGFs) and cytokines, serving to enhance tumorigenesis, foster growth and recruit other bystander cells to sites of metastasis. Through the use of a novel, biomimetic, 3D collagen/hyaluronic acid hydrogel system developed in our labs, this study focused on interactions between primary human macrophages and cancer cells, specifically on how this dialogue impacts the production of the secreted HS-modifying enzymes, heparanase (HPSE) and endosulfatase 1 (SULF1). We studied the impact of co-cultures containing macrophages and endometrial or prostate cancer cell lines, as well as patient-derived xenografts, on the levels of HPSE and SULF1. We also assessed cancer growth characteristics in the presence of macrophages. Taken together, our observations indicate cancer cell responses to macrophages are cell type specific, and we demonstrated significant functional consequences of interactions between macrophages and HS-mediated signaling. We are currently determining if macrophage polarization to classically activated (M1) or alternatively activated (M2) phenotypes alters cancer cell responses in 3D. This system is being used to determine novel approaches to intervene in HBGF delivery to limit cancer growth in a more physiologically relevant in vitro system. Citation Format: Fabio Henrique Brasil da Costa, Nora M. Navone, Mary C. Farach-Carson, Daniel D. Carson. Macrophages alter heparan sulfate-modifying enzyme levels, growth factor activity and cancer cell growth in 3D hydrogels [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 4626. doi:10.1158/1538-7445.AM2017-4626

Abstract 4853: NRP2b, a unique isoform of NRP2, promotes aggressive lung cancer phenotypes

Abstract Neuropilins (NRPs) 1 and 2 are highly-related receptors for class 3 semaphorins, and interact with heparin-binding growth factors and their receptors (e.g. HGF-MET, VEGF-VEGFR2 etc.). We previously reported that NRP2 is upregulated by TGFβ and is required for its pro-tumorigenic activity. Here, we show that this upregulation preferentially involves NRP2b, a largely uninvestigated isoform encoding a divergent, yet highly-conserved C-terminus. Importantly, using a panel of lung cancer cell lines and orthotopic metastasis model, NRP2b promoted migration, invasion, metastasis and tumorsphere formation, whereas the prototype receptor, NRP2a had opposite effects. TGFβ-mediated resistance to gefitinib in EGFR mutated tumors also was dependent on NRP2b expression. In addition, NRP2b, but not NRP2a, linked MET activation by HGF to AKT phosphorylation. Mechanistically, in co-immunoprecipitation assays, we found that NRP2a robustly interacted with PTEN, while the interaction with NRP2b was weak. At the clinical level, NRP2b expression was commonly upregulated in patient lung cancer samples and this upregulation was a significant poor-prognostic factor. Collectively, these data indicate that NRP2b plays an important role in lung cancer invasion, metastasis and EGFR inhibitor resistance. Furthermore, isoform-specific interactions between NRP2 and PTEN may be responsible for the observed differences in MET signaling in response to HGF. Citation Format: Anastasios Dimou, Patrick Nasarre, Joyce Nair-Menon, Federico Cappuzzo, Lorenza Landi, Armida D'Incecco, Hidetaka Uramato, Takeshi Yoshida, Eric Haura, Monica Gooz, Kent Armeson, Robert Gemmill, Harry Drabkin. NRP2b, a unique isoform of NRP2, promotes aggressive lung cancer phenotypes [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 4853. doi:10.1158/1538-7445.AM2017-4853

Targeting Heparin to Collagen within Extracellular Matrix Significantly Reduces Thrombogenicity and Improves Endothelialization of Decellularized Tissues.

Thrombosis within small-diameter vascular grafts limits the development of bioartificial, engineered vascular conduits, especially those derived from extracellular matrix (ECM). Here we describe an easy-to-implement strategy to chemically modify vascular ECM by covalently linking a collagen binding peptide (CBP) to heparin to form a heparin derivative (CBP-heparin) that selectively binds a subset of collagens. Modification of ECM with CBP-heparin leads to increased deposition of functional heparin (by ∼7.2-fold measured by glycosaminoglycan composition) and a corresponding reduction in platelet binding (>70%) and whole blood clotting (>80%) onto the ECM. Furthermore, addition of CBP-heparin to the ECM stabilizes long-term endothelial cell attachment to the lumen of ECM-derived vascular conduits, potentially through recruitment of heparin-binding growth factors that ultimately improve the durability of endothelialization in vitro. Overall, our findings provide a simple yet effective method to increase deposition of functional heparin on the surface of ECM-based vascular grafts and thereby minimize thrombogenicity of decellularized tissue, overcoming a significant challenge in tissue engineering of bioartificial vessels and vascularized organs.

Heparanase regulation of cancer, autophagy and inflammation: new mechanisms and targets for therapy.

Because of its impact on multiple biological pathways, heparanase has emerged as a major regulator of cancer, inflammation and other disease processes. Heparanase accomplishes this by degrading heparan sulfate which regulates the abundance and location of heparin-binding growth factors thereby influencing multiple signaling pathways that control gene expression, syndecan shedding and cell behavior. In addition, heparanase can act via nonenzymatic mechanisms that directly activate signaling at the cell surface. Clinical trials testing heparanase inhibitors as anticancer therapeutics are showing early signs of efficacy in patients further emphasizing the biological importance of this enzyme. This review focuses on recent developments in the field of heparanase regulation of cancer and inflammation, including the impact of heparanase on exosomes and autophagy, and novel mechanisms whereby heparanase regulates tumor metastasis, angiogenesis and chemoresistance. In addition, the ongoing development of heparanase inhibitors and their potential for treating cancer and inflammation are discussed.

The effect of heparinized decellularized scaffolds on angiogenic capability

The immobilization of heparin, a new and versatile approach to the surface modification of decellularized tissues, has the potential to greatly improve the hemocompatibility of engineered tissue constructs derived from decellularized organs. We report on porcine decellularized liver scaffolds (DLSs) heparinized by the end-point attachment (EPA) technique. The heparinized DLSs (HEP-DLSs) have the ability to bind and slowly release heparin-binding growth factors. We hypothesized that DLS-immobilized heparin acts as an antithrombotic coating reagent and binds vascular endothelial growth factor (VEGF) to induce angiogenesis in the DLSs. Human umbilical vein endothelial cells (HUVECs) seeded on HEP-VEGF-DLSs attached and remained bioactive. Using the chicken chorioallantoic membrane (CAM) assay, we found that the HEP-VEGF-DLSs induced a significant and rapid enhancement of angiogenesis compared with native DLSs. Scaffolds were implanted in the greater omentum of rats and evaluated after 7, 14, 21, and 28 days. There were significant increases in the numbers of blood vessels in the HEP-VEGF-DLSs compared with native DLSs at all time-points. The modified method introduced in this article could overcome obstacles faced by conventional matrices that lack the ability to induce rapid and sufficient vascularization. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3021-3030, 2016.

Abstract B22: Necuparanib inhibits pancreatic cancer progression and invasion in a 3D tumor and stromal cell co-culture system

Abstract Pancreatic cancer is one of the most aggressive types of cancer, with only about 5% of patients surviving 5 years past the initial diagnosis. Despite advances with new chemotherapy combinations, overall survival outcomes are still dismal due to the cancer's complex pathology. Novel multi-targeted agents or therapeutic combinations able to disrupt the aggressive fibrotic microenvironment and restrict tumor proliferation are a current focus in the field. Necuparanib (formerly M402) is a heparan sulfate-like molecule that binds and inhibits multiple heparin-binding growth factors, chemokines, and adhesion molecules in cancer progression and metastasis and as such has been shown to modulate multiple pathways in tumors. In order to further explore how necuparanib affects the tumor and its microenvironment, a 3-dimensional (3D) culture system mimicking pancreatic cancer was developed containing a co-culture of human pancreatic tumor cells and pancreatic stellate cells, the most abundant stromal cell in the pancreas. Morphological and histological assessment of the 3D tumor spheroids demonstrated that necuparanib inhibits a) pancreatic tumor cell growth, b) pancreatic tumor cell invasion, and c) pancreatic stellate cell growth. Necuparanib's activity in these assays was shown to be dose-dependent. Moreover, using a negative control for necuparanib engineered to lack the ability to bind growth factors, we established that necuparanib's activity is growth factor binding dependent. The ability to inhibit both tumor and stromal cells demonstrates that necuparanib is a very promising multi-targeting agent for difficult-to-treat cancers such as desmoplastic pancreatic tumors. These studies aid in our understanding of the key biological targets and pathways responsible for necuparanib's anti-tumor effects and support the current Phase 2 safety and efficacy study being performed in patients with metastatic pancreatic cancer. Citation Format: Amanda MacDonald, Michelle Priess, Jennifer Curran, Silva Krause. Necuparanib inhibits pancreatic cancer progression and invasion in a 3D tumor and stromal cell co-culture system. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B22.

Abstract 5102: Heparan sulfate modifying enzymes deliver cancer, stromal and macrophage-produced heparin-binding growth factors in the prostate cancer microenvironment

Abstract We previously showed that the heparan sulfate proteoglycan, perlecan (PLN)/HSPG2, is deposited into the matrix of reactive stroma surrounding tumors, where it concentrates heparin binding growth factors (HBGFs) and cytokines at the tumor-stromal border both in primary prostatic tumors and bone metastatic lesions. Heparan sulfate (HS) modifying enzymes produced in tumor-associated stroma play key roles in the delivery of these perlecan-sequestered cancer, stromal and tumor macrophage-produced HBGFs and cytokines, serving to enhance tumorigenesis and recruit other bystander cells to sites of metastasis. Studies presented here focused on the production of secreted HS-modifying enzyme heparanase and the two extracellular sulfatases, SULF1 and SULF2, in the prostatic tumor microenvironment. Our studies showed that SULF1 and heparanase mRNA levels are elevated in prostate cancer (PCa) cells of the LNCaP lineage in the presence of stroma, indicating that both stroma and PCa cells can be sources of these enzymes. We are presently examining a number of PCa patient derived xenografts to determine the prevalence of SULF-1 and heparanase elevation in castrate resistant disease. To study function, we developed a modified 3D hyaluronate gel system that contains HS-bearing recombinant perlecan domain I (PlnDI) to retain and deliver HBGFs and cytokines to various PCa cells cultured with bone marrow stromal cells, osteoblasts, or tumor macrophages. We are using this system to study the expression and individual functions of heparanase and the two SULFs in promoting HBGF-dependent PCa progression in a physiologically relevant system. Overexpression and gene deletion techniques allow us to determine the role that heparanase or SULFs play in each relevant cell type present in the tumor microenvironment, and thus determine the need for co-targeting of both tumor and reactive stroma. (Supported by NIH P01 and CAPES). Citation Format: Fabio H. Brasil, Valeria Ferrer, Nora M. Navone, Mary C. Farach-Carson, Daniel D. Carson. Heparan sulfate modifying enzymes deliver cancer, stromal and macrophage-produced heparin-binding growth factors in the prostate cancer microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5102.