Laminin fragments conjugated with perlecan's growth factor-binding domain differentiate human induced pluripotent stem cells into skin-derived precursor cells.

Deriving stem cells to regenerate full-thickness human skin is important for treating skin disorders without invasive surgical procedures. Our previous protocol to differentiate human induced pluripotent stem cells (iPSCs) into skin-derived precursor cells (SKPs) as a source of dermal stem cells employs mouse fibroblasts as feeder cells and is therefore unsuitable for clinical use. Herein, we report a feeder-free method for differentiating iPSCs into SKPs by customising culture substrates. We immunohistochemically screened for laminins expressed in dermal papillae (DP) and explored the conditions for inducing the differentiation of iPSCs into SKPs on recombinant laminin E8 (LM-E8) fragments with or without conjugation to domain I of perlecan (PDI), which binds to growth factors through heparan sulphate chains. Several LM-E8 fragments, including those of LM111, 121, 332, 421, 511, and 521, supported iPSC differentiation into SKPs without PDI conjugation. However, the SKP yield was significantly enhanced on PDI-conjugated LM-E8 fragments. SKPs induced on PDI-conjugated LM111-E8 fragments retained the gene expression patterns characteristic of SKPs, as well as the ability to differentiate into adipocytes, osteocytes, and Schwann cells. Thus, PDI-conjugated LM-E8 fragments are promising agents for inducing iPSC differentiation into SKPs in clinical settings.

Structural basis for FGF hormone signalling.

α/ßKlotho coreceptors simultaneously engage fibroblast growth factor (FGF) hormones (FGF19, FGF21 and FGF23)1,2 and their cognate cell-surface FGF receptors (FGFR1-4) thereby stabilizing the endocrine FGF-FGFR complex3-6. However, these hormones still require heparan sulfate (HS) proteoglycan as an additional coreceptor to induce FGFR dimerization/activation and hence elicit their essential metabolic activities6. To reveal the molecular mechanism underpinning the coreceptor role of HS, we solved cryo-electron microscopy structures of three distinct 1:2:1:1 FGF23-FGFR-αKlotho-HS quaternary complexes featuring the 'c' splice isoforms of FGFR1 (FGFR1c), FGFR3 (FGFR3c) or FGFR4 as the receptor component. These structures, supported by cell-based receptor complementation and heterodimerization experiments, reveal that a single HS chain enables FGF23 and its primary FGFR within a 1:1:1 FGF23-FGFR-αKlotho ternary complex to jointly recruit a lone secondary FGFR molecule leading to asymmetric receptor dimerization and activation. However, αKlotho does not directly participate in recruiting the secondary receptor/dimerization. We also show that the asymmetric mode of receptor dimerization is applicable to paracrine FGFs that signal solely in an HS-dependent fashion. Our structural and biochemical data overturn the current symmetric FGFR dimerization paradigm and provide blueprints for rational discovery of modulators of FGF signalling2 as therapeutics for human metabolic diseases and cancer.

Proteoglycans: a common portal for SARS-CoV-2 and extracellular vesicle uptake.

As structural components of the glycocalyx, heparan sulfate proteoglycans (HSPGs) are involved in multiple pathophysiological processes at the apex of cell signaling cascades, and as endocytosis receptors for particle structures, such as lipoproteins, extracellular vesicles, and enveloped viruses, including SARS-CoV-2. Given their diversity and complex biogenesis regulation, HSPGs remain understudied. Here we compile some of the latest studies focusing on HSPGs as internalizing receptors of extracellular vesicles ("endogenous virus") and SARS-CoV-2 lipid-enclosed particles and highlight similarities in their biophysical and structural characteristics. Specifically, the similarities in their biogenesis, size, and lipid composition may explain a common dependence on HSPGs for efficient cell-surface attachment and uptake. We further discuss the relative complexity of extracellular vesicle composition and the viral mechanisms that evolve towards increased infectivity that complicate therapeutic strategies addressing blockade of their uptake.

Coreceptor functions of cell surface heparan sulfate proteoglycans.

Receptor-ligand interactions play an important role in many biological processes by triggering specific cellular responses. These interactions are frequently regulated by coreceptors that facilitate, alter, or inhibit signaling. Coreceptors work in parallel with other specific and accessory molecules to coordinate receptor-ligand interactions. Cell surface heparan sulfate proteoglycans (HSPGs) function as unique coreceptors because they can bind to many ligands and receptors through their HS and core protein motifs. Cell surface HSPGs are typically expressed in abundance of the signaling receptors and, thus, are capable of mediating the initial binding of ligands to the cell surface. HSPG coreceptors do not possess kinase domains or intrinsic enzyme activities and, for the most part, binding to cell surface HSPGs does not directly stimulate intracellular signaling. Because of these features, cell surface HSPGs primarily function as coreceptors for many receptor-ligand interactions. Given that cell surface HSPGs are widely conserved, they likely serve fundamental functions to preserve basic physiological processes. Indeed, cell surface HSPGs can support specific cellular interactions with growth factors, morphogens, chemokines, extracellular matrix (ECM) components, and microbial pathogens and their secreted virulence factors. Through these interactions, HSPG coreceptors regulate cell adhesion, proliferation, migration, and differentiation, and impact the onset, progression, and outcome of pathophysiological processes, such as development, tissue repair, inflammation, infection, and tumorigenesis. This review seeks to provide an overview of the various mechanisms of how cell surface HSPGs function as coreceptors.

Is heparan sulfate a target for inhibition of RNA virus infection?

Heparan sulfate (HS) is a linear polysaccharide attached to a core protein, forming heparan sulfate proteoglycans (HSPGs) that are ubiquitously expressed on the surface of almost all mammalian cells and the extracellular matrix. HS orchestrates the binding of various signal molecules to their receptors, thus regulating many biological processes, including homeostasis, metabolism, and various pathological processes. Due to its wide distribution and negatively charged properties, HS is exploited by many viruses as a cofactor to attach to host cells. Therefore, inhibition of the interaction between virus and HS is proposed as a promising approach to mitigate viral infection, including SARS-CoV-2. In this review, we summarize the interaction manners of HS with viruses with focus on significant pathogenic RNA viruses, including alphaviruses, flaviviruses, and coronaviruses. We also provide an overview of the challenges we may face when using HS mimetics as antivirals for clinical treatment. More studies are needed to provide a further understanding of the interplay between HS and viruses both in vitro and in vivo, which will favor the development of specific antiviral inhibitors.

HIV-1 Tat and Heparan Sulfate Proteoglycans Orchestrate the Setup of in Cis and in Trans Cell-Surface Interactions Functional to Lymphocyte Trans-Endothelial Migration.

HIV-1 transactivating factor Tat is released by infected cells. Extracellular Tat homodimerizes and engages several receptors, including integrins, vascular endothelial growth factor receptor 2 (VEGFR2) and heparan sulfate proteoglycan (HSPG) syndecan-1 expressed on various cells. By means of experimental cell models recapitulating the processes of lymphocyte trans-endothelial migration, here, we demonstrate that upon association with syndecan-1 expressed on lymphocytes, Tat triggers simultaneously the in cis activation of lymphocytes themselves and the in trans activation of endothelial cells (ECs). This "two-way" activation eventually induces lymphocyte adhesion and spreading onto the substrate and vascular endothelial (VE)-cadherin reorganization at the EC junctions, with consequent endothelial permeabilization, leading to an increased extravasation of Tat-presenting lymphocytes. By means of a panel of biochemical activation assays and specific synthetic inhibitors, we demonstrate that during the above-mentioned processes, syndecan-1, integrins, FAK, src and ERK1/2 engagement and activation are needed in the lymphocytes, while VEGFR2, integrin, src and ERK1/2 are needed in the endothelium. In conclusion, the Tat/syndecan-1 complex plays a central role in orchestrating the setup of the various in cis and in trans multimeric complexes at the EC/lymphocyte interface. Thus, by means of computational molecular modelling, docking and dynamics, we also provide a characterization at an atomic level of the binding modes of the Tat/heparin interaction, with heparin herein used as a structural analogue of the heparan sulfate chains of syndecan-1.

Heparan Sulfate Proteoglycans in Viral Infection and Treatment: A Special Focus on SARS-CoV-2.

Heparan sulfate proteoglycans (HSPGs) encompass a group of glycoproteins composed of unbranched negatively charged heparan sulfate (HS) chains covalently attached to a core protein. The complex HSPG biosynthetic machinery generates an extraordinary structural variety of HS chains that enable them to bind a plethora of ligands, including growth factors, morphogens, cytokines, chemokines, enzymes, matrix proteins, and bacterial and viral pathogens. These interactions translate into key regulatory activity of HSPGs on a wide range of cellular processes such as receptor activation and signaling, cytoskeleton assembly, extracellular matrix remodeling, endocytosis, cell-cell crosstalk, and others. Due to their ubiquitous expression within tissues and their large functional repertoire, HSPGs are involved in many physiopathological processes; thus, they have emerged as valuable targets for the therapy of many human diseases. Among their functions, HSPGs assist many viruses in invading host cells at various steps of their life cycle. Viruses utilize HSPGs for the attachment to the host cell, internalization, intracellular trafficking, egress, and spread. Recently, HSPG involvement in the pathogenesis of SARS-CoV-2 infection has been established. Here, we summarize the current knowledge on the molecular mechanisms underlying HSPG/SARS-CoV-2 interaction and downstream effects, and we provide an overview of the HSPG-based therapeutic strategies that could be used to combat such a fearsome virus.

A Systems View of the Heparan Sulfate Interactome.

Heparan sulfate proteoglycans consist of a small family of proteins decorated with one or more covalently attached heparan sulfate glycosaminoglycan chains. These chains have intricate structural patterns based on the position of sulfate groups and uronic acid epimers, which dictate their ability to engage a large repertoire of heparan sulfate-binding proteins, including extracellular matrix proteins, growth factors and morphogens, cytokines and chemokines, apolipoproteins and lipases, adhesion and growth factor receptors, and components of the complement and coagulation system. This review highlights recent progress in the characterization of the so-called "heparan sulfate interactome," with a major focus on systems-wide strategies as a tool for discovery and characterization of this subproteome. In addition, we compiled all heparan sulfate-binding proteins reported in the literature to date and grouped them into a few major functional classes by applying a networking approach.

Dynamics of the lens basement membrane capsule and its interaction with connective tissue-like extracapsular matrix proteins.

The lens, suspended in the middle of the eye by tendon-like ciliary zonule fibers and facing three different compartments of the eye, is enclosed in what has been described as the thickest basement membrane in the body. While the protein components of the capsule have been a subject of study for many years, the dynamics of capsule formation, and the region-specific relationship of its basement membrane components to one another as well as to other matrix molecules remains to be explored. Through high resolution confocal and super-resolution imaging of the lens capsule and 3D surface renderings of acquired z-stacks, our studies revealed that each of its basement membrane proteins, laminin, collagen IV, nidogen and perlecan, has unique structure, organization, and distribution specific both to the region of the lens that the capsule is located in and the position of the capsule within the eye. We provide evidence of basal membrane gradients across the depth of the capsule as well as the synthesis of distinct basement membrane lamella within the capsule. These distinctions are most prominent in the equatorial capsule zone where collagen IV and nidogen span the capsule depth, while laminin and perlecan are located in two separate lamellae located at the innermost and outermost capsule domains. We discovered that an extracapsular matrix compartment rich in the connective tissue-like matrix molecules fibronectin, tenascin-C, and fibrillin is integrated with the superficial surface of the lens capsule. Each matrix protein in this extracapsular zone also exhibits region-specific distribution with fibrils of fibrillin, the matrix protein that forms the backbone of the ciliary zonules, inserting within the laminin/perlecan lamella at the surface of the equatorial lens capsule.

Heparan Sulfate Proteoglycans Biosynthesis and Post Synthesis Mechanisms Combine Few Enzymes and Few Core Proteins to Generate Extensive Structural and Functional Diversity.

Glycosylation is a common and widespread post-translational modification that affects a large majority of proteins. Of these, a small minority, about 20, are specifically modified by the addition of heparan sulfate, a linear polysaccharide from the glycosaminoglycan family. The resulting molecules, heparan sulfate proteoglycans, nevertheless play a fundamental role in most biological functions by interacting with a myriad of proteins. This large functional repertoire stems from the ubiquitous presence of these molecules within the tissue and a tremendous structural variety of the heparan sulfate chains, generated through both biosynthesis and post synthesis mechanisms. The present review focusses on how proteoglycans are "gagosylated" and acquire structural complexity through the concerted action of Golgi-localized biosynthesis enzymes and extracellular modifying enzymes. It examines, in particular, the possibility that these enzymes form complexes of different modes of organization, leading to the synthesis of various oligosaccharide sequences.

Heparin-binding Peptides as Novel Therapies to Stop SARS-CoV-2 Cellular Entry and Infection.

Heparan sulfate proteoglycans (HSPGs) are cell surface receptors that are involved in the cellular uptake of pathologic amyloid proteins and viruses, including the novel coronavirus; severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Heparin and heparan sulfate antagonize the binding of these pathogens to HSPGs and stop their cellular internalization, but the anticoagulant effect of these agents has been limiting their use in the treatment of viral infections. Heparin-binding peptides (HBPs) are suitable nonanticoagulant agents that are capable of antagonizing binding of heparin-binding pathogens to HSPGs. Here, we review and discuss the use of HBPs as viral uptake inhibitors and will address their benefits and limitations to treat viral infections. Furthermore, we will discuss a variant of these peptides that is in the clinic and can be considered as a novel therapy in coronavirus disease 2019 (COVID-19) infection. SIGNIFICANCE STATEMENT: The need to discover treatment modalities for COVID-19 is a necessity, and therapeutic interventions such as heparin-binding peptides (HBPs), which are used for other cases, can be beneficial based on their mechanisms of actions. In this paper, we have discussed the application of HBPs as viral uptake inhibitors in COVID-19 and explained possible mechanisms of actions and the therapeutic effects.

Heparan Sulfate Proteoglycan Signaling in Tumor Microenvironment.

In the last few decades, heparan sulfate (HS) proteoglycans (HSPGs) have been an intriguing subject of study for their complex structural characteristics, their finely regulated biosynthetic machinery, and the wide range of functions they perform in living organisms from development to adulthood. From these studies, key roles of HSPGs in tumor initiation and progression have emerged, so that they are currently being explored as potential biomarkers and therapeutic targets for cancers. The multifaceted nature of HSPG structure/activity translates in their capacity to act either as inhibitors or promoters of tumor growth and invasion depending on the tumor type. Deregulation of HSPGs resulting in malignancy may be due to either their abnormal expression levels or changes in their structure and functions as a result of the altered activity of their biosynthetic or remodeling enzymes. Indeed, in the tumor microenvironment, HSPGs undergo structural alterations, through the shedding of proteoglycan ectodomain from the cell surface or the fragmentation and/or desulfation of HS chains, affecting HSPG function with significant impact on the molecular interactions between cancer cells and their microenvironment, and tumor cell behavior. Here, we overview the structural and functional features of HSPGs and their signaling in the tumor environment which contributes to tumorigenesis and cancer progression.

Design, Synthesis and Bioactive Evaluation of Oxime Derivatives of Dehydrocholic Acid as Anti-Hepatitis B Virus Agents.

Oxime derivatives of dehydrocholic acid and its esters were designed for anti-hepatitis B virus (HBV) drugs according to principles of assembling active chemical fragments. Twelve compounds were synthesized from dehydrocholic acid by esterification and oxime formation, and their anti-hepatitis B virus (HBV) activities were evaluated with HepG 2.2.15 cells. Results showed that 5 compounds exhibited more effective inhibition of HBeAg than positive control, among them 2b-3 and 2b-1 showed significant anti-HBV activities on inhibiting secretion of HBeAg (IC50 (2b-3) = 49.39 ± 12.78 µM, SI (2b-3) = 11.03; IC50 (2b-1) = 96.64 ± 28.99 µM, SI (2b-1) = 10.35) compared to the Entecavir (IC50 = 161.24 µM, SI = 3.72). Molecular docking studies showed that most of these compounds interacted with protein residues of heparan sulfate proteoglycan (HSPG) in host hepatocyte and bile acid receptor.

Syndecan-1 Facilitates the Human Mesenchymal Stem Cell Osteo-Adipogenic Balance.

Bone marrow-derived human mesenchymal stems cells (hMSCs) are precursors to adipocyte and osteoblast lineage cells. Dysregulation of the osteo-adipogenic balance has been implicated in pathological conditions involving bone loss. Heparan sulfate proteoglycans (HSPGs) such as cell membrane-bound syndecans (SDCs) and glypicans (GPCs) mediate hMSC lineage differentiation and with syndecan-1 (SDC-1) reported in both adipogenesis and osteogenesis, these macromolecules are potential regulators of the osteo-adipogenic balance. Here, we disrupted the HSPG profile in primary hMSC cultures via temporal knockdown (KD) of SDC-1 using RNA interference (RNAi) in undifferentiated, osteogenic and adipogenic differentiated hMSCs. SDC-1 KD cultures were examined for osteogenic and adipogenic lineage markers along with changes in HSPG profile and common signalling pathways implicated in hMSC lineage fate. Undifferentiated hMSC SDC-1 KD cultures exhibited a pro-adipogenic phenotype with subsequent osteogenic differentiation demonstrating enhanced maturation of osteoblasts. In cultures where SDC-1 KD was performed following initiation of differentiation, increased adipogenic gene and protein marker expression along with increased Oil Red O staining identified enhanced adipogenesis, with impaired osteogenesis also observed in these cultures. These findings implicate SDC-1 as a facilitator of the hMSC osteo-adipogenic balance during early induction of lineage differentiation.

An investigation of genetic polymorphisms in heparan sulfate proteoglycan core proteins and key modification enzymes in an Australian Caucasian multiple sclerosis population.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease affecting the central nervous system in young adults. Heparan sulfate proteoglycans (HSPGs) are ubiquitous to the cell surface and the extracellular matrix. HSPG biosynthesis is a complex process involving enzymatic attachment of heparan sulfate (HS) chains to a core protein. HS side chains mediate specific ligand and growth factor interactions directing cellular processes including cell adhesion, migration and differentiation. Two main families of HSPGs exist, the syndecans (SDC1-4) and glypicans (GPC1-6). The SDCs are transmembrane proteins, while the GPC family are GPI linked to the cell surface. SDC1 has well-documented interactions with numerous signalling pathways. Genome-wide association studies (GWAS) have identified regions of the genome associated with MS including a region on chromosome 13 containing GPC5 and GPC6. International studies have revealed significant associations between this region and disease development. The exostosin-1 (EXT1) and sulfatase-1 (SULF1) are key enzymes contributing to the generation of HS chains. EXT1, with documented tumour suppressor properties, is involved in the initiation and polymerisation of the growing HS chain. SULF1 removes 6-O-sulfate groups from HS chains, affecting protein-ligand interactions and subsequent downstream signalling with HS modification potentially having significant effects on MS progression. In this study, we identified significant associations between single nucleotide polymorphisms in SDC1, GPC5 and GPC6 and MS in an Australian Caucasian case-control population. Further significant associations in these genes were identified when the population was stratified by sex and disease subtype. No association was found for EXT1 or SULF1.

Heparan Sulfate Proteoglycans and Viral Attachment: True Receptors or Adaptation Bias?

Heparan sulfate proteoglycans (HSPG) are composed of unbranched, negatively charged heparan sulfate (HS) polysaccharides attached to a variety of cell surface or extracellular matrix proteins. Widely expressed, they mediate many biological activities, including angiogenesis, blood coagulation, developmental processes, and cell homeostasis. HSPG are highly sulfated and broadly used by a range of pathogens, especially viruses, to attach to the cell surface.

Perlecan domain I gradients establish stable biomimetic heparin binding growth factor gradients for cell migration in hydrogels.

Growth factor gradients orchestrate many biological processes including organogenesis, wound healing, cancer invasion, and metastasis. Heparin-binding growth factor (HBGF) gradients are established in living systems by proteoglycans including the extracellular matrix heparan sulfate proteoglycan, perlecan/HSPG2. Three potential HBGF-binding glycosaminoglycan attachment sites occur in N-terminal domain I of perlecan's five domains. Our overarching goal was to form stable, biomimetic non-covalently bound HBGF gradients surrounding cells encapsulated in hyaluronate-based hydrogels by first establishing perlecan domain I (PlnD1) gradients. A versatile multichannel gradient maker device (MGMD) was designed and 3D printed, then used to create desired gradients of microparticles in hydrogels. Next, we used the device to covalently incorporate gradients of PEGylated PlnD1 in hydrogels with high-low-high or high-medium-low concentrations across the hydrogel width. Fluorescently-labeled fibroblast growth factor-2 was delivered to hydrogels in phosphate-buffered saline and allowed to electrostatically bind to the covalently pre-incorporated PlnD1, producing stable non-covalent HBGF gradients. To test cell viability after flow through the MGMD, delicate primary human salivary stem/progenitor cells were encapsulated in gradient hydrogels where they showed high viability and continued to grow. Next, to test migratory behavior in response to HBGF gradients, two cell types, preosteoblastic MC3T3-E1 cell line and breast cancer cell line MDA-MB-231 were encapsulated in or adjacent to PlnD1-modified hydrogels. Both cell lines migrated toward HBGFs bound to PlnD1. We conclude that establishing covalently-bound PlnD1 gradients in hydrogels provides a new means to establish physiologically-relevant gradients of HBGFs that are useful for a variety of applications in tissue engineering and cancer biology. STATEMENT OF SIGNIFICANCE: Gradients of heparin binding growth factors (HBGFs) direct cell behavior in living systems. HBGFs bind electrostatically to gradients of HS proteoglycans in the extracellular matrix creating HBGF gradients. We recreated HBGF gradients in physiological hyaluronate-based hydrogels using a 3D-printed multichannel gradient maker device (MGMD) that created gradients of HS proteoglycan-derived perlecan/HSPG2 domain I. We demonstrated the ability of a variety of cells, including primary salivary stem/progenitor cells, pre-osteoblastic cells and an invasive breast cancer cell line, to be co-encapsulated in gradient hydrogels by flowing them together through the MGMD. The versatile device and the ability to create HBGF gradients in hydrogels for a variety of applications is innovative and of broad utility in both cancer biology and tissue engineering applications.

Pathogenic effects of agrin V1727F mutation are isoform specific and decrease its expression and affinity for HSPGs and LRP4.

Agrin is a large extracellular matrix protein whose isoforms differ in their tissue distribution and function. Motoneuron-derived y+z+ agrin regulates the formation of the neuromuscular junction (NMJ), while y-z- agrin is widely expressed and has diverse functions. Previously we identified a missense mutation (V1727F) in the second laminin globular (LG2) domain of agrin that causes severe congenital myasthenic syndrome. Here, we define pathogenic effects of the agrin V1727F mutation that account for the profound dysfunction of the NMJ. First, by expressing agrin variants in heterologous cells, we show that the V1727F mutation reduces the secretion of y+z+ agrin compared to wild type, whereas it has no effect on the secretion of y-z- agrin. Second, we find that the V1727F mutation significantly impairs binding of y+z+ agrin to both heparin and the low-density lipoprotein receptor-related protein 4 (LRP4) coreceptor. Third, molecular modeling of the LG2 domain suggests that the V1727F mutation primarily disrupts the y splice insert, and consistent with this we find that it partially occludes the contribution of the y splice insert to agrin binding to heparin and LRP4. Together, these findings identify several pathogenic effects of the V1727F mutation that reduce its expression and ability to bind heparan sulfate proteoglycan and LRP4 coreceptors involved in the muscle-specific kinase signaling pathway. These defects primarily impair the function of neural y+z+ agrin and combine to cause a severe CMS phenotype, whereas y-z- agrin function in other tissues appears preserved.

The Limitations of Collagen/CPP Hybrid Peptides as Carriers for Cancer Drugs to FaDu Cells.

The in vitro efficacy of cancer prodrugs varies significantly between malignant cell lines. The most commonly identified problems relate to delivery: uptake mechanism, endosomal entrapment, and drug release. Here we present the study of collagen/cell penetrating hybrid (COL/CPP) peptide carriers intended to deliver paclitaxel to the hypopharyngeal carcinoma (FaDu) cells. Confocal microscopy imaging revealed the surprising response of FaDu cell to COL/CPP in comparison to previously studied cancer cell lines: hybrid peptides that carry both COL and CPP domain adsorb on the FaDu cell surface. While the CPP domain was design to facilitate the cellular uptake, in the case of FaDu cells, it also induced detrimental interactions with the cell membrane. Despite surface adsorption, the colocalization study with endosomal markers EEA1 and LAMP1 reveals that COL/CPP is internalized via endosomal pathway, peptides are able to escape before lysosome formation and release paclitaxel. Therefore, the main obstacle for paclitaxel delivery to FaDu cells appears to be related to cell surface properties. This behavior seems specific to FaDu cells, and could be linked to previously reported overexpression of T5, heparanase splice variants that produces protein lacking enzymatic activity of heparanase. This results in increased concentration of HSPG on FaDu cell surface, and possibly creates a barrier for cellular uptake of highly charged COL/CPP.

TRF2 positively regulates SULF2 expression increasing VEGF-A release and activity in tumor microenvironment.

The telomeric protein TRF2 is overexpressed in several human malignancies and contributes to tumorigenesis even though the molecular mechanism is not completely understood. By using a high-throughput approach based on the multiplexed Luminex X-MAP technology, we demonstrated that TRF2 dramatically affects VEGF-A level in the secretome of cancer cells, promoting endothelial cell-differentiation and angiogenesis. The pro-angiogenic effect of TRF2 is independent from its role in telomere capping. Instead, TRF2 binding to a distal regulatory element promotes the expression of SULF2, an endoglucosamine-6-sulfatase that impairs the VEGF-A association to the plasma membrane by inducing post-synthetic modification of heparan sulfate proteoglycans (HSPGs). Finally, we addressed the clinical relevance of our findings showing that TRF2/SULF2 expression is a worse prognostic biomarker in colorectal cancer (CRC) patients.