Geometrical microfeature cues for directing tubulogenesis of endothelial cells

Angiogenesis, the formation of new blood vessels by sprouting from pre-existing ones, is critical for the establishment and maintenance of complex tissues. Angiogenesis is usually triggered by soluble growth factors such as VEGF. However, geometrical cues also play an important role in this process. Here we report the induction of angiogenesis solely by SVVYGLR peptide micropatterning on polymer surfaces. SVVYGLR peptide stripes were micropatterned onto polymer surfaces by photolithography to study their effects on endothelial cell (EC) behavior. Our results showed that the EC behaviors (cell spreading, orientation and migration) were significantly more guided and regulated on narrower SVVYGLR micropatterns (10 and 50 μm) than on larger stripes (100 μm). Also, EC morphogenesis into tube formation was switched on onto the smaller patterns. We illustrated that the central lumen of tubular structures can be formed by only one-to-four cells due to geometrical constraints on the micropatterns which mediated cell-substrate adhesion and generated a correct maturation of adherens junctions. In addition, sprouting of ECs and vascular networks were also induced by geometrical cues on surfaces micropatterned with SVVYGLR peptides. These micropatterned surfaces provide opportunities for mimicking angiogenesis by peptide modification instead of exogenous growth factors. The organization of ECs into tubular structures and the induction of sprouting angiogenesis are important towards the fabrication of vascularized tissues, and this work has great potential applications in tissue engineering and tissue regeneration.     

Synthetic extracellular matrices for in situ tissue engineering

Cell interactions with the extracellular matrix play important roles in guiding tissue morphogenesis. The matrix stimulates cells to influence such things as differentiation and the cells actively remodel the matrix via local proteolytic activity. We have designed synthetic hydrogel networks that participate in this interplay: They signal cells via bound adhesion and growth factors, and they also respond to the remodeling influence of cell-associated proteases. Poly(ethylene glycol)-bis-vinylsulfone was crosslinked by a Michael-type addition reaction with a peptide containing three cysteine residues, the peptide sequence being cleavable between each cysteine residue by the cell-associated protease plasmin. Cells were able to invade gel networks that contained adhesion peptides and were crosslinked by plasmin-sensitive peptides, while materials lacking either of these two characteristics resisted cell infiltration. Incorporated bone morphogenetic protein-2 (BMP-2) induced bone healing in a rat model in materials that were both adhesive and plasmin-sensitive, while materials lacking plasmin sensitivity resisted formation of bone within the material. Furthermore, when a heparin bridge was incorporated as a BMP-2 affinity site, mimicking yet another characteristic of the extracellular matrix, statistically improved bone regeneration was observed.     

Identification of heparin affin regulatory peptide domains with potential role on angiogenesis

Heparin affin regulatory peptide (HARP) is a growth factor displaying high affinity for heparin. It is present in the extracellular matrix of many tissues, interacting with heparan sulfate and dermatan/chondroitin sulfate glycosaminoglycans. We have previously shown that HARP is implicated in the control of angiogenesis and its effects are mimicked, at least in part, by synthetic peptides that correspond to its N and C termini. In the present work, we show that HARP is cleaved by plasmin, leading to the production of five peptides that correspond to distinct domains of the molecule. Heparin, heparan sulfate and dermatan sulfate, at various HARP to glycosaminoglycan ratios, partially protect HARP from plasmin degradation. The molecules with higher affinity to HARP are the more protective, heparin being the most efficient. The peptides that are produced from cleavage of HARP by plasmin, affect in vivo and in vitro angiogenesis and modulate the angiogenic activity of vascular endothelial growth factor on human umbilical vein endothelial cells. Similar results were obtained in vitro with recombinant HARP peptides, identical to the peptides generated after treatment of HARP with plasmin. These results suggest that different regions of HARP may induce or inhibit angiogenesis.     

Biochemical and biophysical aspects of collagen nanostructure in the extracellular matrix

ECM is composed of different collagenous and non-collagenous proteins. Collagen nanofibers play a dominant role in maintaining the biological and structural integrity of various tissues and organs, including bone, skin, tendon, blood vessels, and cartilage. Artificial collagen nanofibers are increasingly significant in numerous tissue engineering applications and seem to be ideal scaffolds for cell growth and proliferation. The modern tissue engineering task is to develop three-dimensional scaffolds of appropriate biological and biomechanical properties, at the same time mimicking the natural extracellular matrix and promoting tissue regeneration. Furthermore, it should be biodegradable, bioresorbable and non-inflammatory, should provide sufficient nutrient supply and have appropriate viscoelasticity and strength. Attributed to collagen features mentioned above, collagen fibers represent an obvious appropriate material for tissue engineering scaffolds. The aim of this minireview is, besides encapsulation of the basic biochemical and biophysical properties of collagen, to summarize the most promising modern methods and technologies for production of collagen nanofibers and scaffolds for artificial tissue development.     

Selectively desulfated heparin inhibits fibroblast growth factor-induced mitogenicity and angiogenesis

Fibroblast growth factors (FGFs) are known to induce formation of new blood vessels, angiogenesis. We show that FGF-induced angiogenesis can be modulated using selectively desulfated heparin. Chinese hamster ovary cells (CHO677) deficient in heparan sulfate biosynthesis were employed to assess the function of heparin/heparan sulfate in FGF receptor-1 (FGFR-1) signal transduction and biological responses. In the presence of FGF-2, FGFR-1 kinase and subsequent mitogen-activated protein kinase Erk2 activities were augmented in a dose-dependent manner, whereas high concentrations of heparin resulted in decreased activity. The length of the heparin oligomer, minimally an 8/10-mer, was critical for the ability to enhance FGFR-1 kinase activity. The N- and 2-O-sulfate groups of heparin were essential for binding to FGF-2, whereas stimulation of FGFR-1 and Erk2 kinases by FGF-2 also required the presence of 6-O-sulfate groups. Sulfation at 2-O- and 6-O-positions was moreover a prerequisite for binding of heparin to a lysine-rich peptide corresponding to amino acids 160-177 in the extracellular domain of FGFR-1. Selectively 6-O-desulfated heparin, which binds to FGF-2 but fails to bind the receptor, decreased FGF-2-induced proliferation of CHO677 cells, presumably by displacing intact heparin. Furthermore, FGF-2-induced angiogenesis in chick embryos was inhibited by 6-O-desulfated heparin. Thus, formation of a ternary complex of FGF-2, heparin, and FGFR-1 appears critical for the activation of FGFR-1 kinase and downstream signal transduction. Preventing complex formation by modified heparin preparations may allow regulation of FGF-2 functions, such as induction of angiogenesis.     

Incorporation of heparin-binding peptides into fibrin gels enhances neurite extension: an example of designer matrices in tissue engineering.

The goal of this work was to improve the potential of fibrin to promote nerve regeneration by enzymatically incorporating exogenous neurite-promoting heparin-binding peptides. The effects on neurite extension of four different heparin-binding peptides, derived from the heparin-binding domains of antithrombin III, neural cell adhesion molecule and platelet factor 4, were determined. These exogenous peptides were synthesized as bi-domain peptide chimeras, with the second domain being a substrate for factor XIIIa. This coagulation transglutaminase covalently bound the peptides within the fibrin gel during coagulation. The heparin-binding peptides enhanced the degree of neurite extension from embryonic chick dorsal root ganglia through 3-dimensional fibrin gels, and the extent of enhancement was found to correlate positively with the heparin-binding affinity of the individual domains. The enhancement could be inhibited by competition with soluble heparin, by degradation of cell-surface proteoglycans, and by inhibition of the covalent immobilization of the peptide. These results demonstrate an important potential role for proteoglycan-binding components of the extracellular matrix in neurite extension and suggest that fibrin gels modified with covalently bound heparin-binding peptides could serve as a therapeutic agent to enhance peripheral nerve regeneration through nerve guide tubes. More generally, the results demonstrate that the biological responses to fibrin, the body's natural wound healing matrix, can be dramatically improved by the addition of exogenous bioactive peptides in a manner such that they become immobilized during coagulation.-Sakiyama, S. E., Schense, J. C., Hubbell, J. A. Incorporation of heparin-binding peptides into fibrin gels enhances neurite extension: an example of designer matrices in tissue engineering.     

Induction of vascular smooth muscle cell growth by selective activation of the thrombin receptor. Effect of heparin.

The synthetic peptide, SFLLRNPNDKYEPF, has been recently described as a peptide mimicking the new amino-terminus created by cleavage of the thrombin receptor, therefore acting as an agonist of the thrombin receptor. This peptide was a potent mitogen for rabbit arterial smooth muscle cells (SMC) and exhibited the same activity as that of native alpha-thrombin. Both compounds stimulated the proliferation of growth-arrested SMCs with half-maximum mitogenic responses at 1 nM. NAPAP, a synthetic inhibitor of the enzymatic activity of thrombin, specifically inhibited thrombin-induced SMC growth (IC50 = 0.35 +/- 0.04 microM) but was without effect on the mitogenic effect of the agonist peptide. These results therefore demonstrate that the mitogenic effect of alpha-thrombin for SMCs is intimately linked to its esterolytic activity. Heparin, which inhibited fetal calf serum-induced SMC growth, was without effect on thrombin-induced SMC growth but strongly reduced the mitogenic effect of the agonist peptide (IC50 = 32 +/- 5 micrograms/ml). This effect was not related to the anti-coagulant activity of heparin but was highly dependent on molecular mass and on the global charge of the molecule and was also observed for other sulphated polysaccharides such as pentosan polysulphate.     

Specificities of heparin-binding sites from the amino-terminus and type 1 repeats of thrombospondin-1

Interactions of heparin with intact human thrombospondin-1 (TSP1) and with two heparin-binding fragments of TSP1 were characterized using chemically modified heparins, a vascular heparan sulfate proteoglycan, and a series of heparin oligosaccharides prepared by partial deaminative cleavage. The avidity of TSP1 binding increased with oligosaccharide size, with plateaus at 4 to 6 and at 8 to 10 monosaccharide units. The dependence on oligosaccharide size for binding to the recombinant amino-terminal heparin-binding domain of TSP1 was the same as that of the intact TSP1 molecule but differed from that of a synthetic heparin-binding peptide from the type 1 repeats, suggesting that the interaction between intact TSP1 and heparin is primarily mediated by the amino-terminal domain. Based on activities of chemically modified heparins, binding to TSP1 depended primarily on 2-N- and 6-O-sulfation of glucosamine and to a lesser degree on 2,3-O-sulfation and the carboxyl residues of the uronic acids. In contrast, all of these modifications were required for binding of heparin to the type 1 repeat peptides. Affinity purification of heparin octasaccharides on immobilized TSP1 type 1 repeat peptides revealed a preference for oligosaccharides containing the disaccharide sequence IdoA(2-OSO(3))alpha1-4-GlcNS(6-OSO(3)). Binding of these oligosaccharides to the peptide required the Trp residues. These data demonstrate that the heparin-binding specificities of intact TSP1 and peptides from the type 1 repeats overlap with that of basic fibroblast growth factor (FGF2) and are consistent with the ability of these TSP1-derived molecules to inhibit FGF2-stimulated angiogenesis.     

Identification of a heparin binding domain of the neural cell adhesion molecule N-CAM using synthetic peptides

The neural cell adhesion molecule (N-CAM) plays an integral role in cell interactions during neural development, with the binding of heparan sulfate proteoglycan to the amino-terminal region of N-CAM being required for N-CAM function. In the present study we have used synthetic peptides (HBD-1 and HBD-2), derived from the primary amino acid sequence of rat N-CAM, to identify the region of N-CAM that binds heparan sulfate. The 28 amino acid HBD-1 synthetic peptide was shown to bind both [3H]heparin and dissociated retinal cells. Retinal cells also attach to a substratum of HBD-2 peptide, but fail to bind to a control peptide containing a scrambled amino acid sequence of HBD-2. The HBD-2 peptide also inhibits retinal cell adhesion to N-CAM, demonstrating the physiological importance of the amino acid sequence encoded by the HBD peptide. These data therefore permit the localization of a heparin binding domain to a 17 amino acid region of immunoglobulin-like loop 2.     

Regulation and function of connective tissue growth factor/CCN2 in tissue repair, scarring and fibrosis

Connective tissue growth factor (CTGF, CCN2) is a secreted protein with major roles in angiogenesis, chondrogenesis, osteogenesis, tissue repair, cancer and fibrosis. It is a member of the CCN family of immediate-early gene products which are characterised by four discrete protein modules in which reside growth factor binding domains, functional motifs for integrin recognition, heparin and proteoglycan binding, and dimerization motifs. A primary function of CTGF is to modulate and coordinate signaling responses involving cell surface proteoglycans, key components of the extracellular matrix, and growth factors. Integration of these molecular cues regulates growth factor and receptor interactions, cell motility and mesenchymal cell activation and differentiation in tissue remodelling. Abnormal amplification of CTGF dependent signals results in a failure to terminate tissue repair, leading pathological scarring in conditions such as fibrosis and cancer.     

Direct and allosteric inhibition of the FGF2/HSPGs/FGFR1 ternary complex formation by an antiangiogenic, thrombospondin-1-mimic small molecule

Fibroblast growth factors (FGFs) are recognized targets for the development of therapies against angiogenesis-driven diseases, including cancer. The formation of a ternary complex with the transmembrane tyrosine kinase receptors (FGFRs), and heparan sulphate proteoglycans (HSPGs) is required for FGF2 pro-angiogenic activity. Here by using a combination of techniques including Nuclear Magnetic Resonance, Molecular Dynamics, Surface Plasmon Resonance and cell-based binding assays we clarify the molecular mechanism of inhibition of an angiostatic small molecule, sm27, mimicking the endogenous inhibitor of angiogenesis, thrombospondin-1. NMR and MD data demonstrate that sm27 engages the heparin-binding site of FGF2 and induces long-range dynamics perturbations along FGF2/FGFR1 interface regions. The functional consequence of the inhibitor binding is an impaired FGF2 interaction with both its receptors, as demonstrated by SPR and cell-based binding assays. We propose that sm27 antiangiogenic activity is based on a twofold-direct and allosteric-mechanism, inhibiting FGF2 binding to both its receptors.     

Glycosaminoglycans mimetics potentiate the clonogenicity, proliferation, migration and differentiation properties of rat mesenchymal stem cells

Successful use of stem cell-based therapeutic products is conditioned by transplantation of optimized cells in permissive microenvironment. Mesenchymal stem cell (MSC) fates are tightly regulated by humoral factors, cellular interactions and extracellular matrix (ECM) components, such as glycosaminoglycans (GAG), which are complex polysaccharides with structural heterogeneity. During osteogenesis, a temporally controlled expression of particular GAG species is required to interact with specific growth promoting and differentiating factors to regulate their biological activities. As a comparative tool to study natural GAG, we used structurally and functionally related synthetic GAG mimetics. One of these compounds [OTR₄₁₂₀] was previously shown to stimulate bone repair in rat models. Here, we demonstrate that structurally distinct GAG mimetics stimulate differentially clonogenicity, proliferation, migration and osteogenic phenotype of MSC in vitro, according to their specific chemical signature, underlying the role of sulfate and acetyl groups in specific interactions with heparin binding factors (HBF). These effects are dependent on FGF-2 interactions since they are inhibited by a FGF receptor 1 signaling pathway blocker. These data suggest that the in vivo [OTR₄₁₂₀] bone regenerative effect could be due to its ability to induce MSC migration and osteogenic differentiation. To conclude, we provide evidences showing that GAG mimetics may have great interest for bone regeneration therapy and represent an alternative to exogenous growth factor treatments to optimize potential therapeutic properties of MSC.     

Angiogenic activity of osteopontin-derived peptide SVVYGLR

Angiogenesis plays an important role in various pathological conditions as well as some physiological processes. Although a number of soluble angiogenic factors have been reported, extracellular matrix also has crucial effect on angiogenesis through interaction with endothelial cells. Since recent reports showed osteopontin had some angiogenic activity, the effect of the SVVYGLR peptide, novel binding motif in osteopontin molecule, on angiogenesis was examined in this study. Synthetic peptide SVVYGLR did not have proliferative effect on endothelial cells but adhesion and migration activity to endothelial cells. Furthermore, SVVYGLR had as potent activity for tube formation in three-dimensional collagen gel as vascular endothelial growth factor which is known to be the strongest angiogenic factor. Electron microscopical analysis showed a number of microvilli on the endothelial luminar surface and tight junction formation in the luminar intercellular border between endothelial cells, indicating SVVYGLR induced cell porarity and differentiation of endothelial cells. This small peptide might be expected to stimulate angiogenesis to improve some ischemic conditions in the future because of some advantages due to smaller molecular weight.     

A substituted dextran enhances muscle fiber survival and regeneration in ischemic and denervated rat EDL muscle.

Ischemia and denervation of EDL muscle of adult rat induce a large central zone of degeneration surrounded by a thin zone of peripheral surviving muscle fibers. Muscle regeneration is a complex phenomenon in which many agents interact, such as growth factors and heparan sulfate components of the extracellular matrix. We have shown that synthetic polymers, called RGTA (as regenerating agents), which imitate the heparan sulfates, are able to stimulate tissue repair when applied at the site of injury. In crushed muscles, RGTA were found to accelerate both regeneration and reinnervation. In vitro, RGTA act as protectors and potentiators of various heparin binding growth factors (HBGF). It was postulated that in vivo their tissue repair properties were due in part to an increase of bioavailability of endogenously released HBGF. In the present work, we show that ischemic and denervated EDL muscle treated by a unique injection of RGTA differs from the control after 1 wk in several aspects: 1) the epimysial postinflammatory reaction is inhibited and the area of fibrotic tissue among fibers is reduced; 2) the peripheral zone, as measured by the number of intact muscle fibers, was increased by more than twofold; and 3) In the central zone, RGTA enhances the regeneration of the muscle fibers as well as muscle revascularization. These results suggest that RGTA both protects muscle fibers from degeneration and preserves the differentiated state of the surviving fibers. For the first time it is demonstrated that a functionalized polymeric compound can prevent some of the damage resulting from muscle ischemia. RGTA may therefore open a new therapeutic approach for muscle fibrosis and other postischemic muscle pathologies.     

Electrospun synthetic and natural nanofibers for regenerative medicine and stem cells

Nanofibers are attractive substrates for tissue regeneration applications because they structurally mimic the native extracellular matrix. Electrospinning has been recognized as one of the most efficient techniques to fabricate polymer nanofibers. Recent research has demonstrated that cellular responses, for example attachment, proliferation and differentiation, can be modulated by tuning nanofiber properties. In combination with other processing techniques, such as particulate leaching or three-dimensional printing, nanofibrous scaffolds incorporating macroporous networks could be developed to enhance infiltration of cells. Three dimensional nanofiber-based constructs offer an opportunity to achieve advanced functional tissue regeneration. This review explores the advantageous effects of nanofibers on cell behaviors compared to traditional scaffolds.     

Alkaline phosphatase dual‐binding sites for collagen dictate cell migration and microvessel assembly in vitro

Interactions between cell types, growth factors, and extracellular matrix components involved in angiogenesis are crucial for new vessel formation leading to tissue regeneration. This study investigated whether cocultures of fibroblasts and endothelial cells (ECs; from macro‐ or microvasculature) play a role in the formation of microvessel‐like structures by ECs, as well as modulate fibroblast differentiation and growth factors production (vascular endothelial cell growth factor, basic fibroblast growth factor, active transforming growth factor‐β1, and interleukin‐8), which are important for vessel sprouting and maturation. Data obtained revealed that in vitro coculture systems of fibroblasts and human ECs stimulate collagen synthesis and growth factors production by fibroblasts that ultimately affect the formation and distribution of microvessel‐like structures in cell cultures. In this study, areas with activated fibroblasts and high alkaline phosphatase (ALP) activity were also observed in cocultures. Molecular docking assays revealed that ALP has two binding positions for collagen, suggesting its impact in collagen proteins’ aggregation, cell migration, and microvessel assembly. These findings indicate that bioinformatics and coculture systems are complementary tools for investigating the participation of proteins, like collagen and ALP in angiogenesis.     

A Peptide Derived from the HIV-1 gp120 Coreceptor-Binding Region Promotes Formation of PAP248-286 Amyloid Fibrils to Enhance HIV-1 Infection

BackgroundSemen is a major vehicle for HIV transmission. Prostatic acid phosphatase (PAP) fragments, such as PAP248-286, in human semen can form amyloid fibrils to enhance HIV infection. Other endogenous or exogenous factors present during sexual intercourse have also been reported to promote the formation of seminal amyloid fibrils.

Methodology and Principal Findings

Here, we demonstrated that a synthetic 15-residue peptide derived from the HIV-1 gp120 coreceptor-binding region, designated enhancing peptide 2 (EP2), can rapidly self-assemble into nanofibers. These EP2-derivated nanofibers promptly accelerated the formation of semen amyloid fibrils by PAP248-286, as shown by Thioflavin T (ThT) and Congo red assays. The amyloid fibrils presented similar morphology, assessed via transmission electron microscopy (TEM), in the presence or absence of EP2. Circular dichroism (CD) spectroscopy revealed that EP2 accelerates PAP248-286 amyloid fibril formation by promoting the structural transition of PAP248-286 from a random coil into a cross-β-sheet. Newly formed semen amyloid fibrils effectively enhanced HIV-1 infection in TZM-bl cells and U87 cells by promoting the binding of HIV-1 virions to target cells.

Conclusions and Significance

Nanofibers composed of EP2 promote the formation of PAP248-286 amyloid fibrils and enhance HIV-1 infection.     

The fibroblast growth factor-binding protein FGF-BP

Fibroblast growth factors (FGFs) are important regulators of cell migration, proliferation and differentiation, e.g., during embryogenesis and wound healing, and under several pathological conditions including tumor growth and tumor angiogenesis. Since heparin-binding FGFs are tightly bound to heparansulfate proteoglycans, and therefore, trapped in the extracellular matrix, their release through the action of an FGF-binding protein (FGF-BP) is one of the critical steps in FGF bioactivation. FGF-BP expression is highly tissue specific and strictly regulated through different promoter elements. Besides its role in embryogenesis and wound healing, FGF-BP is upregulated in several tumors and it is associated especially with early stages of tumor formation, where angiogenesis plays a critical role. Concomitantly, in several mouse tumor models, targeting of FGF-BP by ribozymes or RNA interference (RNAi) abolishes or reduces tumor growth and tumor angiogenesis. This indicates that FGF-BP can be rate-limiting for tumor growth and serves as an angiogenic switch molecule, and that it represents an increasingly promising target molecule in anti-tumor therapy.