WO2018053166A2 - Matériaux de stimulation de l'intégrine pour la normalisation d'un système vasculaire malade - Google Patents

Matériaux de stimulation de l'intégrine pour la normalisation d'un système vasculaire malade Download PDF

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WO2018053166A2
WO2018053166A2 PCT/US2017/051616 US2017051616W WO2018053166A2 WO 2018053166 A2 WO2018053166 A2 WO 2018053166A2 US 2017051616 W US2017051616 W US 2017051616W WO 2018053166 A2 WO2018053166 A2 WO 2018053166A2
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integrin
fibronectin
polypeptide
vegf
hydrogel
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WO2018053166A3 (fr
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Tatiana Segura
Thomas H. Barker
Shuoran LI
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The Regents Of The University Of California
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6903—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel

Definitions

  • the invention relates to methods and materials useful for vascular patterning including vascular morphology control and VEGF permeability reduction.
  • therapeutic angiogenic materials to treat cardiovascular diseases, such as deficient blood supply to the heart, limbs, and brain, has primarily been driven by the delivery of angiogenic factors within a scaffold. Optimization of these materials has been focused dominantly on controlling angiogenic factor release or presentation and modulating bulk physical properties. Although adhesive ligands that promote integrin binding are generally incorporated within therapeutic angiogenic materials, the subsequent cell-material interaction has not been explored as an angiogenic signal.
  • Integrins are family of heterodimeric transmembrane proteins that mediate cell surface binding to the extracellular matrix (ECM) and intracellular actin cytoskeletal components (see, e.g. 1) (numbers in this text such as " 1" represent the articles cited in the reference section at the end of the specification). These receptors are formed by pairs of alpha (a) and beta ( ⁇ ) subunits, and have been associated with processes ranging from cell structure and adhesion to cell differentiation and survival, which are cell behaviors critical to tissue morphogenesis, homeostasis and repair (see, e.g. 1-3).
  • At least seven a and ⁇ heterodimers are expressed by endothelial cells and have been implicated in vascular morphogenesis and vessel patterning (see, e.g. 4-6). Though a complete understanding role of all these integrin pairs in the process of angiogenesis is yet to be available, the role of ⁇ and ⁇ 3 integrins in angiogenesis is important in vascular lumen formation (see, e.g. 7, 8) and tight cell-cell junction formation (see, e.g. 7, 9-13).
  • both up-regulation and abolishment of ⁇ and ⁇ 3 integrin activation have shown to be related to pathological angiogenesis (see, e.g. 1, 11-16).
  • pathological angiogenesis see, e.g. 1, 11-16.
  • ⁇ integrin a psoriasis phenotype
  • the knockout of ⁇ integrin resulted in the weakening of endothelial cell junctions and induction of blood leakage in a retinal angiogenesis assay (see, e.g. 11).
  • the upregulation ⁇ 3 integrin leads to enhanced endothelial cell permeability (see, e.g.
  • one main function of the ECM during the angiogenesis process is to present the growing vessels with the appropriate integrin binding ligands to generate normal, non-pathological vessels.
  • Materials designed for therapeutic angiogenesis should likewise, provide the appropriate integrin binding ligands to support revascularization of diseased tissues.
  • Integrin binding peptides derived from natural ECM proteins is a popular approach to promote integrin engagement (see, e.g. 18-23).
  • Integrin-binding RGD peptide derived from fibronectin is by far the most widely utilized peptide in the generation of materials for cell growth in vitro or promote tissue repair in vivo.
  • integrin-binding peptides can support cell attachment, migration, and differentiation, they have severely reduced binding affinity and specificity compared to the same peptide presented within 3D structure of the full-length protein.
  • the expression process is difficult and an alternative approach to present peptide motifs in a native 3D structural context is needed.
  • fibronectin (Fn) fragments of the 9th type III repeat and 10th type III repeat (Fn 1119-10) have been expressed to present RGD sequence in the correct 3D structural context and improve binding affinity and specificity (see, e.g. 24, 25).
  • Fn recombinant fibronectin
  • these protein fragments do not contain the natural switches that modulate integrin engagement (e.g. native fibronectin can bind several integrin pairs depending on the level of extension of the protein) (see, e.g. 26-29) and, thus, lack complete specificity.
  • the fibronectin 9th type III repeat and 10th type III repeat has been engineered to promote ⁇ 3 ⁇ 1 and ⁇ 5 ⁇ 1 (" ⁇ 3/ ⁇ 5 ⁇ 1") integrin heterodimer specific binding and shown to enhance bone formation, mesenchymal stem cell differentiation toward bone, and modulate epithelial to mesenchymal transition.
  • the extracellular matrix (ECM) has also been successfully engineered to coordinate and modulate simultaneous integrin and growth factor signaling to enhance vascularization, bone formation, and skin healing.
  • ⁇ 3/ ⁇ 5 ⁇ 1 or ⁇ 3 integrin heterodimers together with engineered VEGF delivery can be used to modulate endothelial cell physiology in a manner that generates differential vascular patterns within matrices.
  • certain fibronectin polypeptides can preferentially engage ⁇ 3/ ⁇ 5 ⁇ 1, and alternatively, certain fibronectin polypeptides can preferentially engage ⁇ 3 integrin heterodimers.
  • the selective engagement of ⁇ 3/ ⁇ 5 ⁇ 1 integrin heterodimers modulates endothelial cell physiology in a manner that results in vascular patterning having an enhanced vessel penetration, density and maturity as compared to the engagement of different integrin heterodimers (such as the ⁇ 3 integrin heterodimer).
  • This discovery can be harnessed to selectively modulate vascular patterning in vitro and in vivo.
  • the invention disclosed herein has a number of embodiments including methods for selectively engaging ⁇ 3/ ⁇ 5 ⁇ 1 or ⁇ 3 integrin heterodimers in a manner that modulates endothelial cell physiology as well as compositions for use in these methods.
  • Embodiments of the invention include, for example, methods of using the fibronectin polypeptides disclosed herein to normalize diseased vasculature.
  • This work shows that precisely controlled integrin activation from a biomaterial can be harnessed to direct therapeutic vessel regeneration and reduce VEGF induced vascular permeability in vivo.
  • the invention disclosed herein shows that specific integrin activation from a biomaterial can be harnessed to direct vascular patterning in vitro and in vivo, vascular patterning which result in enhanced reperfusion of the brain after trauma such as stroke.
  • integrin stimulation from engineered matrices is a morphogenic signal that can be harnessed to generate either a normal vasculature or a diseased vasculature depending on the integrin being engaged by the fibronectin polypeptide.
  • Figures la-i show that HUVEC sprouting is greatly affected by integrin activation.
  • Figures lb and lc Representative immunofluorescent images for sprouting in both Fibl and Fib3 fibrin gel. Scale bar: 100 ⁇ .
  • Figures lb and lc Quantification of sprouts number and branch points in Fib3 gels.
  • Figures Id and le Quantification of filopodia per tip in Fib3 gels.
  • Figure If Comparison of sprouts number between Fibl and Fib3 gels.
  • Figures lg and lh Quantification of sprouts number and branch points in Fibl gels.
  • Figure li Representative filopodia image in Fibl gel. Scale bar: 50 um. *,**,*** and **** indicate P ⁇ 0.05, P ⁇ 0.01, P ⁇ 0.001 and P ⁇ 0.0001, respectively.
  • Figures 2a-g show that intra-joint and intra-loop structure exists in gels dosed with 9(4G)10 and RGD.
  • Figure 2a Comparison of branch structure between 9(4G)10 and 9* 10 conditions. Scale bar: 50 ⁇ .
  • Figure 2b Intra-loop and intra- joints structures in Fib3 gel with 1000 ⁇ RGD. Scale bar: 50 ⁇ .
  • Figure 2c Branch cluster occurrence comparison between Fib3 gels.
  • Figures 2d and 2e Branch cluster occurrence and sprouts number quantifications of 0,200,500, 1000 ⁇ RGD Fib3 gels.
  • FIG. 2f Microscopic analysis of whole bead sprouting effects integrin (scale bar: 100 ⁇ ) and intra-joint and intra-loop branch structures (scale bar: 50 um) from blockage of ⁇ , a5, ⁇ or ⁇ 3 on 2 ⁇ Fn9* 10 or 2 ⁇ Fn9(4G)10 Fibl gels.
  • Figures 3a-i show that microscopic analysis of EGFP-HUVEC sprouting assay anastomosis at day 11.
  • Figures 3a, 3d, and 3g Inter-beads branch overview of blank, 2 ⁇ Fn9* 10 and 2 ⁇ Fn9(4G)10 Fibl gels. Scale bar: 200 um.
  • Figures 3b, 3e, and 3h Sample 1 of bead-bead interactions. Scale bar: 100 ⁇ .
  • Figures 3c, 3f, and 3i Sample 2 of bead-bead interactions. Scale bar: 100 ⁇ .
  • Figure 4a-d show VE-cadherin analysis both on 2D surfaces and in 3D fibrin gels.
  • Figures 4a-4c Microscopic analysis and quantification of 2D VE-cadherin distribution on fragment coated cell culture dish without VEGF dosage after 12 hours. Scale bar: 50 ⁇ . ** indicate P ⁇ 0.01.
  • Figure 4d Microscopic analysis of VE- cadherin signals from blockage of av integrin on 2 ⁇ Fn9(4G)10 Fiblgels in comparison to 2 ⁇ Fn9* 10 Fibl gels. Scale bar: 100 ⁇ (whole bead) and 50 ⁇ (sprouts).
  • Figure 5a-g show data from a matrigel plug assay
  • Figure 5a Scheme for synthesizing 100% L, 75% L, 50% L and 25% L VEGF nanocapsules.
  • Figure 5b Scheme for HA Hydrogels containing fibronectin fragments and equal amount of each type of VEGF nanocapsules.
  • Figure 5c Comparison of vessel morphologies among normal mice skin, the surfaces of blank and 10 ⁇ fragment-loaded HA hydrogels. Scale bar: 200 ⁇ .
  • Figure 5d Vessel tortuosity comparison between 9* 10 and 9(4G)10 conditions. Scale bar: 50 ⁇ .
  • Figure 5e 3D heat map view for vessel penetration visualization.
  • Figures 5f and 5g Projection view and quantification for vessel penetration distance. Scale bar: 100 ⁇ . ** and **** indicate P ⁇ 0.01 and P ⁇ 0.0001, respectively.
  • Figure 6a shows a schematic illustration of mouse brain coronal sections showing a cortical stroke and the transplantation of an injectable hyaluronic acid (HA) hydrogel within the damaged area represented by the asterisk.
  • HA hyaluronic acid
  • This stroke cavity is situated directly adjacent to the region of the brain that undergoes the most substantial repair and recovery, the peri-infarct tissue, where new structures such as vessels and axons develop and infiltrate the infarct while undergoing a drastic remodeling and leakiness.
  • the growing vasculature structure and permeability are associated with tissue repair.
  • Figure 6b shows fluorescent microscopy showing brain vasculature in both the infarct and peri-infarct (stained for Glut-1 or Glut-1 plus tomato lectin intravascular perfusion) as well as leaked red blood cells (stained for Ter-119) in the different conditions. Scale bar: 100 ⁇ .
  • Figure 6c shows that the results show a significantly increased positive area for stained vessels in both the infarct and the per-infarct.
  • Figure 6d shows that areas of nV+star transplanted mice compared with any other group. ** and *** indicate P ⁇ 0.01 and P ⁇ 0.001, respectively.
  • Figure 6e shows that the measure of Ter-119 positive red blood cells in the two nV conditions show a significantly reduced area in the infarct site of nV+star transplanted mice compared with nV+4G. This result shows that nV+star decreased leakiness of growing vessels in the stroke brain. * indicates P ⁇ 0.05.
  • Figure 6f shows that the morphoanalysis of growing vessels in the peri-infarct area shows a significantly increased number of vascular ramifications in the nV+star condition compared with the nV+4G, suggesting that nV+star promotes a post-stoke vascular remodeling into a more physiological network. * indicates P ⁇ 0.05.
  • the invention disclosed herein has a number of embodiments.
  • On embodiment of the invention is a method of using a fibronectin polypeptide to preferentially bind one or more integrin heterodimers within a population of integrin heterodimers that includes ⁇ 3 integrin heterodimers and ⁇ 3/ ⁇ 5 ⁇ 1 integrin heterodimers.
  • the method comprises selecting a fibronectin polypeptide comprising SEQ ID NO: 1 to preferentially bind o3l ⁇ 5 ⁇ 1 integrin heterodimers within the population of integrin heterodimers; or selecting a fibronectin polypeptide comprising SEQ ID NO: 2 to preferentially bind ⁇ 3 integrin heterodimers within the population of integrin heterodimers.
  • the polypeptide of SEQ ID NO: 1 or the polypeptide SEQ ID NO: 2 is combined with the population of integrin heterodimers so that they preferentially bind ⁇ 3 integrin heterodimers or ⁇ 3/ ⁇ 5 ⁇ 1 integrin heterodimers within the population of integrin heterodimers.
  • the fibronectin polypeptide of SEQ ID NO: 1 or the polypeptide SEQ ID NO: 2 is covalently coupled to a hydrogel composition.
  • a hydrogel composition comprising SEQ ID NO: 1 or SEQ ID NO: 2 coupled to a heterologous amino acid sequence (e.g. a protease recognition sequence, a histidine tag etc.).
  • the hydrogel is crosslinked by degradable crosslinkers such as protease degradable peptides.
  • the hydrogel composition further comprises human vascular endothelial growth factor (VEGF) having the amino acid sequence shown in SEQ ID NO: 3.
  • VEGF human vascular endothelial growth factor
  • the VEGF is disposed within a polymer nanocapsule having polymers crosslinked by crosslinking agents selected to degrade within an in vivo environment so as to control the release of the VEGF from the composition into the in vivo environment.
  • the crosslinking agents selected to degrade within an in vivo environment comprise protease degradable peptides formed from D and L amino acids.
  • the population of integrin polypeptides is disposed on the surface of a vascular endothelial cell and the fibronectin polypeptide is combined with the vascular endothelial cells such that the binding of the fibronectin polypeptide modulates vascular endothelial cell physiology.
  • binding of the fibronectin polypeptide to the vascular endothelial cell modulates at least one of endothelial cell vessel sprouting and/or filopodia development in endothelial tip cells (well-known endothelial cell physiological phenomena as discussed for example in DeSmet et al., Arterioscler Thromb Vase Biol. 2009 May;29(5):639-49; Eiken et al., Curr Opin Cell Biol. 2010 Oct;22(5):617-25; and Eelen et al., Trends Endocrinol Metab. 2013 Dec;24(12):589- 96).
  • a related embodiment of the invention is a method of modulating vessel sprouting in human endothelial cells by combining the endothelial cells (e.g. endothelial cells disposed in a wound or site of trauma) with a composition comprising a hydrogel (e.g. a hyaluronic acid hydrogel), a fibronectin polypeptide coupled to the hydrogel which comprises a 9th type III repeat of fibronectin (Fn III9) and a 10th type III repeat of fibronectin (Fn III10); and human vascular endothelial growth factor (VEGF) comprising the amino acids in SEQ ID NO: 3.
  • a hydrogel e.g. a hyaluronic acid hydrogel
  • a fibronectin polypeptide coupled to the hydrogel which comprises a 9th type III repeat of fibronectin (Fn III9) and a 10th type III repeat of fibronectin (Fn III10)
  • VEGF human vascular endothelial growth factor
  • Fn III9 comprises SEQ ID NO: 4
  • Fn III10 comprises SEQ ID NO: 5 and the polypeptide preferentially binds ⁇ 3 ⁇ 1 and/or ⁇ 5 ⁇ 1 integrin heterodimers.
  • the fibronectin polypeptide is coupled to a heterologous amino acid sequence such as one comprising a protease recognition site. This method comprises allowing the fibronectin polypeptide to bind to ⁇ 3/ ⁇ 5 ⁇ 1 integrin heterodimers expressed by the endothelial cells and allowing the VEGF to bind to VEGF receptors on the endothelial cells so that vessel sprouting and growth in the endothelial cells is modulated.
  • the VEGF is disposed within a polymer nanocapsule having polymers crosslinked by crosslinking agents comprising peptide selected to degrade within an in vivo environment so as to control the release of the VEGF from the composition into the in vivo environment.
  • crosslinking agents comprise peptides comprise selected amounts of D and L amino acids.
  • Another embodiment of the invention is a composition of matter comprising a polypeptide having a 9th type III repeat of fibronectin (Fn III9) and a 10th type III repeat of fibronectin (Fn III10).
  • Fn III9 comprises SEQ ID NO: 4
  • Fn III10 comprises SEQ ID NO: 5
  • Fn III9 and Fn III10 are linked together by a heterologous amino acid linker comprising at least two amino acid residues (e.g. an amino acid linker comprising two to five glycine residues); and the polypeptide preferentially binds avP3-integrin as compared to ⁇ 5 ⁇ 1 integrin.
  • Certain embodiments comprise avP3-integrin and ⁇ 5 ⁇ 1 -integrin, with amounts of ⁇ 3- integrin bound to the polypeptide being greater than amounts of a5pi-integrin or ⁇ 3 ⁇ 1 -integrin bound to the polypeptide.
  • Certain embodiments further comprise a hydrogel covalently coupled to the polypeptide.
  • this hydrogel is crosslinked by a degradable moiety such as protease degradable peptides.
  • Yet another embodiment of the invention is a composition of matter comprising a hyaluronic acid hydrogel crosslinked by protease degradable peptides, human vascular endothelial growth factor (VEGF) comprising the amino acids in SEQ ID NO: 3 disposed within a polymer nanocapsule having polymers crosslinked by crosslinking agents comprising peptide selected to degrade within an in vivo environment so as to control the release of the VEGF from the composition into the in vivo environment, wherein said peptides comprise selected amounts of D and L amino acids.
  • VEGF vascular endothelial growth factor
  • composition further includes a polypeptide covalently coupled to the hydrogel comprising a 9th type III repeat of fibronectin (Fn III9) and a 10th type III repeat of fibronectin (Fn IIIIO), Fn III9 and the polypeptide preferentially binds ⁇ 3/ ⁇ 5 ⁇ 1 integrin heterodimers as compared to avP3-integrin heterodimers.
  • a polypeptide covalently coupled to the hydrogel comprising a 9th type III repeat of fibronectin (Fn III9) and a 10th type III repeat of fibronectin (Fn IIIIO), Fn III9 and the polypeptide preferentially binds ⁇ 3/ ⁇ 5 ⁇ 1 integrin heterodimers as compared to avP3-integrin heterodimers.
  • Fibronectin fragments with tunable integrin binding are discussed below.
  • Recombinant fibronectin fragments of the 9th type III repeat (Fn III9) and 10th type III repeat (Fn III10) were designed to preferentially bind ⁇ 3/ ⁇ 5 ⁇ 1 or ⁇ 3 integrin heterodimers respectively. This was achieved by first increasing the thermodynamic stability of Fn III9 through a leucine to proline point mutation at position 1408. This mutation has been previously shown to stabilize the integrin- binding domain of fibronectin, i.e. Fn 1119-10, and enhance its binding selectivity to synergy-dependent ⁇ integrins, including ⁇ 5 ⁇ 1 and ⁇ 3 ⁇ 124, (see, e.g. 29, 41).
  • Self-assembled monolayers on gold were used to specifically immobilize fibronectin fragments and the modified surfaces were used for in vitro characterization.
  • Amine containing self-assembled monolayers were constructed and used to immobilize malemide-modified heparin via carbodiimide chemistry.
  • Fn9* 10 and Fn9(4G)10 were subsequently covalently bound using Michael type addition between the malemide on the surface and the thiol on the N-terminus of the fibronectin fragment.
  • the amount of attached fragments was then quantified by enzyme-linked immunosorbent assay (ELISA) and shown to be the same ( ⁇ 50ng fragment/cm 2 ) for both fragments, indicating that the reactivity of both fragments is similar.
  • ELISA enzyme-linked immunosorbent assay
  • endothelial cells (EC) cultured on fragment-modified surfaces was assessed 24 or 48 hours post plating. ECs were able to attach and spread on either fragment-modified surface. As expected, only ECs seeded on Fn9(4G)10 surfaces showed a positive staining for ⁇ 3, validating that the fragment Fn9* 10 does not mediate significant binding through ⁇ 3.
  • the actin cytoskeleton for cells cultured on Fn9(4G)10 surfaces showed more short and disoriented actin fibers compared with Fn9* 10 surfaces, where actin fibers showed extensive length.
  • VEGF vascular endothelial growth factor A 165
  • Fn9* 10 or Fn9(4G)10 Exposure of ECs plated on Fn9* 10 or Fn9(4G)10 did not change the binding to ⁇ 3, which remained positive for Fn9(4G)10 but not Fn9* 10. Although no proliferation difference was shown for all the conditions tested, EC migration was significantly increased for cells cultured on Fn9(4G)10 modified surfaces. Together these findings confirm that ECs alter their cellular behavior depending on the integrin binding specificity dominating their attachment to the surface.
  • Integrin stimulation guides endothelial cell sprouting patterns
  • fibronectin mediated cell adhesion on vascular endothelial growth factor A 165 (VEGF) induced vascular sprouting.
  • VEGF vascular endothelial growth factor A 165
  • two types of fibrin were used, one that contains fibronectin (Fibl) and one that is fibronectin depleted (Fib3).
  • EC coated beads were suspended in the fibrin matrices and cultured in the presence of 2ng/ml soluble VEGF for 7-days following the protocol of Huges et al (see, e.g. 45, 46). At day 7, the cultures were fixed, stained for actin, and quantified for the number of sprouts and number of branching points per bead.
  • fibronectin-depleted matrices (Fib3) was significantly decreased compared with fibronectin containing matrices (Fib 1, Fig. la), providing evidence that the presence of native fibronectin is critical for EC sprouting.
  • Addition of exogenous fibronectin to Fib3 matrices rescues EC sprouting (p ⁇ 0.005, Fig. lb), demonstrating a strong correlation between fibronectin-cell interactions and EC sprouting.
  • the level of sprouting was significantly lower than that observed in Fib 1 matrices (Fig. If), indicating that the specific fibronectin concentration within the matrix or other factors removed in the Fib3 preparation (e.g. Von Willebrand factor) may also be important for EC sprouting in fibrin.
  • Tip cells are necessary for EC branch formation and lead cells in sprouting branches. They are characterized by the presence of extended filopodia structures, membrane protrusions that extend from the cell and attach to the ECM substrate through integrins (see, e.g. 47). Interestingly, introduction of Fn9* 10, but not 9(4G)10, into both Fib 1 and Fib 3 fibrin matrices showed increased number of filopodia per tip cell (Fig. l d,e,i). Thus, although the introduction of 9(4G)10 fragment into both Fibl and Fib3 increased the number of branch points per bead (Fig. 1 c, g), the number of filopodia in the 9(4G)10 condition was not increased.
  • Upregulation of ⁇ 3 and alterations in its activation state has been associated with disease states such as cancer (see, e.g. 50-52) and fibrosis (see, e.g. 53, 54) and has been widely used as a cancer targeting ligand in drug delivery applications (see, e.g. 55, 56), yet RGD is the most widely used integrin binding peptide to modify biomaterials.
  • Our results show a dose dependent effect of RGD on vascular patterning with increasing doses leading to increased pathological vessels resulting in sprouting vessel clusters.
  • RGD modified biomaterials for therapeutic angiogenesis is inherently flawed; rather, we believe that the incorporation conditions for RGD peptides such as presentation, concentration, and other neighboring ligands should be studied to ensure that the desired revascularization pattern is obtained. For example, clustering RGD within hydrogels has been shown to upregulate the expression of ⁇ integrin (see, e.g. 57) and immobilization of VEGF leads to ⁇ recruitment (see, e.g. 58).
  • Integrin stimulation guides vascular anastomosis
  • Vessel anastomosis is a crucial step in vasculature renewal and repair, guiding the fusion of adjacent vessel branches.
  • the majority of endothelial cells become quiescent, among which only 0.01% still divide (see, e.g. 47).
  • sprouts from parental vessels fuse with other sprouts or pre-existing blood vessels for the purposes of supplying blood and oxygen to surrounding tissues (see, e.g. 59-61). This anastomosis process not only affects vascular network distribution, but also has great impacts on structure, quality and maturation of newly formed vessels.
  • EC bead sprouts were monitored daily and analyzed at day 11 when anastomosis between adjacent beads started. Normal anastomosis results in the binding of tip cells through a single tip cell contact (see, e.g. 62). Clear single tip-tip contact or paralleled tip interaction were observed in both blank and Fn9* 10 conditions (Fig. 3), indicating that further inducing ⁇ 3/ ⁇ 5 ⁇ 1 integrin engagement supports similar anastomosis as native fibronectin present in the Fibl matrix.
  • EGFP enhanced green fluorescent protein
  • VE-cadherin As an important cell-cell junction protein, VE-cadherin is not only responsible for shifting endothelial cell response to VEGF from proliferation and migration to survival and quiescence (see, e.g. 63), but also functions to maintain low permeability of endothelial cell layer (see, e.g. 17). Even partial knockout of VE-cadherin can lead to vascular instability and hemorrhages (see, e.g. 64). Most importantly, VE-cadherin function can be disrupted by upregulation of ⁇ 3 integrin, enhancing endothelial cell permeability (see, e.g. 17). Thus, we hypothesize that ⁇ 3 activating scaffolds lead to pathological intra- vessel and inter-vessel features through VE-cadherin disruption.
  • EC sprouting in Fn9(4G)10 modified fibrin matrices was characterized by greatly reduced VE-cadherin staining on sprout shunts and cell-cell junctions compared with EC sprouting in fibronectin Fn9* 10 modified matrices (Fig. 4d).
  • av integrin binding was disrupted using function- blocking antibodies.
  • VE-cadherin staining after av blocking in Fn9(4G)10 modified fibrin matrices showed EC cells with increased VE-cadherin staining similar to what was observed in Fn9* 10 modified matrices, indicating that av binding is responsible for the reduction in VE-cadherin expression.
  • the effect of av blocking was observed in both Fibl and Fib3 matrices (Fig.4d).
  • Integrin stimulation from a bioengineered matrix guides vascular patterns in vivo
  • HA hydrogel is chosen for our studies because it does not interact with cells through integrin receptors and provides a clean system to study integrin-mediated events.
  • HA hydrogel has been injected in vivo, has been shown to support delivery of biocues and is also currently used under clinical settings.
  • HA hydrogels are formed through crosslinking HA molecules using Michael type addition chemistry between acrylamide groups introduced to the backbone of hyaluronic acid and dithiol crosslinker containing protease degradable peptides (see, e.g. 65).
  • Fn fragments were also introduced to this protease degradable hydrogel matrix backbone to mediate integrin binding using the same Michael type chemistry through the cysteine in the fragment N-terminus.
  • VEGF was incorporated into the system using a controlled release system based on single protein nanocapsules previously developed in our laboratory (see, e.g. 66, 67). Nanocapsules are formed through in situ radical polymerization of acrylate and acrylamide containing monomers and peptide crosslinkers around a protein core. The final product is a protein complex in which the protein is surrounded by a hydrated protease-degradable polymeric shell.
  • Hydrogels containing none or 10 ⁇ fibronectin fragments, 200ng VEGF nanocapsules, and having a storage modulus of 350Pa were implanted subcutaneously (Fig. 5b). Evaluation of isolectin perfused whole mount sections was performed 14-day s after implantation on light sheet microscopy and confocal microscopy. HA hydrogels that do not contain fibronectin fragments (blk) resulted in the least vessel sprouting on the hydrogel surface and vessel infiltration within the hydrogel compared with fragment conditions even with the presence of VEGF nanocapsules, demonstrating that integrin binding is essential for angiogenesis to occur in vivo.
  • blk fibronectin fragments
  • HA hydrogels modified with either fibronectin fragment supported an angiogenic response; however, the morphology of the vessels was significantly different.
  • Fn9* 10 displayed non-tortuous vessels displaying similar features as the normal mouse vasculature (control) while Fn9(4G)10 displayed tortuous and unorganized vessels that appeared to clump with one another (Fig. 5c,d).
  • VEGF is the key regulator of angiogenesis and it has been widely investigated in clinical and preclinical models to promote perfusion in various organ systems (see, e.g. 69-72).
  • VEGF has been plagued with negative clinical trials showing little therapeutic benefit at safe doses (see, e.g. 73-76) and the generation of a leaky and immature vasculature (see, e.g. 77, 78).
  • effective VEGF delivery is a holy grail in the field of therapeutic angiogenesis.
  • VEGF is one of the essential molecules in normal post-stroke angiogenesis (see, e.g.
  • mice were submitted to a cerebral artery occlusion (MCAo) and transplanted 5 days later with a 350Pa HA-RGD hydrogel containing 200ng of VEGF nanocapsules and 10 ⁇ fibronectin fragments (nV+Fn9(4G)10 and nV+Fn9* 10 directly into the stroke cavity (Fig. 6a).
  • Sections were all stained for Glut-1, a glucose transporter expressed on brain endothelial cells and the positively stained vascular area was quantified in both the infarct and peri-infarct areas (Fig. 6b).
  • Glut-1 stained only or Gut-1 plus tomato lectin in tomato lectin-perfused animals were quantified.
  • Tomato lectin alters Glut-1 staining such that in tomato lectin- perfused animals the combination of both stains reveals the vascular bed the same as Glut-1 alone in tomato lectin-unperfused animals.
  • all the VEGF containing hydrogels showed a greater vasculature area percentage than RGD only gels in the infarct and the peri-infarct regions.
  • vascular area was significantly increased in the nV+star condition compared with any other group (Fig.6c, d), providing evidence for a strong role of activated ⁇ 3/ ⁇ 5 ⁇ 1 integrin binding in promoting the angiogenesis process.
  • the significantly increased vessel area percentage of nV+ Fn9* 10 when comparing with Vs+ Fn9* 10 condition in both areas also verified the greater therapeutic effects from VEGF nanocapsules.
  • Ter-119 a red blood cell marker
  • Fig. 6b The results show a significantly reduced positive area for Ter- 119 in the nV+ Fn9* 10 condition compared with the nV+Fn9(4G)10 group, suggesting a beneficial effect of the activation of ⁇ 3/ ⁇ 5 ⁇ 1 in promoting vascular permeability and stability while reducing blood leakage (Fig. 6e).
  • the morphoanalysis of tomato lectin-perfused vessels was performed by quantifying the number of vascular ramification growing out of a common vascular tree.
  • Integrin-specific scaffold for therapeutic angiogenesis In the work presented here, we establish an integrin-specific material platform via the immobilization of synthetic integrin ligands on the basis of an integrin-inert biocompatible material to induce specific ⁇ 3/ ⁇ 5 ⁇ 1 or ⁇ 3 integrin activation.
  • ⁇ 3/ ⁇ 5 ⁇ 1 and ⁇ 3 integrin-specific materials include RGD
  • ⁇ 3 integrin-specific materials lead to pathological tumor-like sprouting clumps, which can later be rescued by the blockage of av integrin.
  • ⁇ 3/ ⁇ 5 ⁇ 1 integrin not only affects vascular patterning in vivo by reducing vessel tortuosity and increasing infiltration distance, but also promotes the development and maturation of newly formed vessels in the damaged brain, thus representing a promising candidate in the design of therapeutic pro-angiogenic scaffolds.
  • Our unique hydrogel platform featuring both controlled growth factor delivery and precise integrin-specificity presents a novel and feasible template for the future design of therapeutic angiogenic scaffolds.
  • Standard laboratory microscope glass slides were sequentially washed with acetone, isopropyl alcohol and methanol before gold deposition in e-beam evaporator.
  • Deposition parameter 5nm titanium at 0.3 A/s deposition rate, followed by 30 nm gold at 0.5 A/s deposition rate.
  • Gold slides were then functionalized with 1% HS- Cl l-EG6- H2(l l-Mercaptoundecyl)hexa(ethylene glycol) amine, ProChimia Surfaces) and 99% HS-C11-EG4-OH (l l-Mercaptoundecyl)tetra(ethylene glycol), Sigma-Aldrich).
  • EMCH N-[e-Maleimidocaproic acid]hydrazide, Fisher Scientific, PI-22106
  • DMSO Dimethyl sulfoxide
  • 5 mg/ml Heparin (Alfa Aesar, A16198) solution in lOOmM 2-(N-morpholino)ethanesulfonic acid (MES) pH6 buffer was then mixed with EMCH, NHS (N-Hydroxysuccinimide , Sigma-Aldrich) and EDC (l-(3- Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, Fisher Scientific) sequentially.
  • Modified gold surfaces was Argon-dried and then assembled together with PDMS sheet that has two 8mm circular wells followed by 60ul/well 0.1%BSA-PBS as blocking buffer for 1 hour at room temperature. After aspiration, 60ul/well of Anti- Fibronectin primary antibody (1 :2000 dilution in blocking buffer, ab299, Abeam) was added for 2 hours at room temperature. After 3 washes using 0.05% Tween-20+PBS (washing buffer), 60ul/well of streptavidin-URP (1 :5000 dilution in blocking buffer, #DY998, R&D Systems) was added for lhour at room temperature.
  • TMB substrate #7004L, Cell signaling
  • Primary antibodies were prepared as follows in blocking buffer: Rabbit anti-mouse and human VEGFR-2 (Cell Signaling Technology; #2479L) - 1 :200, Mouse anti-human PECAM-1 (R&D; #BBA7) - 1 :200, Monoclonal mouse anti-Vinculin antibody (Sigma-Aldrich, #V9131) - 1 :400, Mouse anti-avP3 antibody (EMD Millipore, MAB1976) - 1 :200. Samples were incubated with primary antibodies overnight at 4°C, followed by Secondary antibodies (1 :500) and 2 ⁇ g/ml DAPI for 1 hour in the dark at room temperature. Imaging was performed using a Zeiss confocal and images were analyzed using Image J.
  • Fibrin bead assay HUVEC were mixed with dextran-coated Cytodex 3 microcarriers (Amersham Pharmacia Biotech) at a concentration of 400 HUVEC per bead in 1 ml of EGM-2 medium (Clonetics). Beads with cells were shaken gently every 20 min for 4h at 37°C and 5% C02. After incubating, beads with cells were transferred to a 25-cm2 tissue culture flask (BD Biosciences) and left for 12-16 h in 5 ml of EGM-2 at 37°C and 5% C02.
  • beads with cells were collected and washed three times with 1 ml of EGM-2 w/o Fibronectin and resuspended at a concentration of 500 beads/ml in 2 mg/ml fibrinogen (Fibl or Fib3),
  • Sprouting assay was performed as previously described in Fib3 fibrin gels with 200, 500 or 1000 ⁇ of a 2 PIi-8-RGD (H-NQEQVSPLRGDSPG- H2, SEQ ID NO: 9, GenScript).
  • EGFP-HUVEC were mixed with dextran-coated Cytodex 3 microcarriers at a concentration of 400 HUVEC per bead in 1 ml of EGM-2 medium. Beads with cells were shaken gently every 20 min for 4h at 37°C and 5% C02. Beads with cells were then transferred to a 25-cm2 tissue culture flask and left for 12-16 h in 5 ml of EGM-
  • Fibrinogen/ HUVEC bead/ HDF cells solution was allowed to clot for 5 min at room temperature and then at 37°C and 5% CO2 for 20 min.
  • EGM-2 w/o Fibronectin was added to each well and equilibrated with the fibrin clot for 30 min at 37°C and 5% CO2.
  • Medium was removed from the well and replaced with 1 ml of fresh EGM-2 w/o Fibronectin and later was changed every other day. Bead assays were monitored for 11 days.
  • HUVEC beads were suspended at a concentration of 500 beads/ml in 2 mg/ml fibrinogen (Fibl or Fib3), 1 U /ml factor XIII, 0.04 U/ml aprotinin and 2 ⁇ (high dosage) or 0.267 ⁇ (low dosage) Fn9* 10 at a pH of 7.4 with or without 5 ⁇ g/ml of ⁇ integrin blocking antibody (AIIB2, Developmental Studies Hybridoma Bank) or a5 integrin blocking antibody (BIIG2, Developmental Studies Hybridoma Bank). The blocking antibody ⁇ g/ml) in fresh fibronectin-free EGM-2 medium was replenished every day.
  • Fn9(4G)10 gels HUVEC beads were suspended at a concentration of 500 beads/ml in 2 mg/ml fibrinogen (Fibl or Fib3), 1 U /ml factor XIII, 0.04 U/ml aprotinin and 2 ⁇ (high dosage) or 0.239 ⁇ (low dosage) Fn9(4G)10 at a pH of 7.4 with or without 5 ⁇ g/ml of ⁇ 3 integrin blocking antibody (9H5, Developmental Studies Hybridoma Bank) or av integrin blocking antibody (P3G8, Developmental Studies Hybridoma Bank). The blocking antibody ⁇ g/ml) in fresh fibronectin-free EGM-2 medium was replenished every day.
  • hyaluronan was modified to contain acrylate functionalities. Briefly, hyaluronic acid (2.0 g, 5.28 mmol, 60 kDa) was reacted with 18.0 g (105.5 mmol) of adipic acid dihydrazide (ADH) at pH 4.75 in the presence of 4.0 g (20 mmol) of 1- ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride overnight and purified through dialysis (8000 MWCO) against a 100-0 mM salt gradient water for 2 days. The purified intermediate (HA-ADH) was lyophilized and stored at -20 °C until used.
  • ADH adipic acid dihydrazide
  • HA-ADH (1.9 g) was reacted with N-acryloxysuccinimide (NHS-Ac) (1.33 g, 4.4 mmol) in HEPES buffer (10 mM HEPES, 150 mM NaCl, 10 mM EDTA, pH 7.2) overnight and purified through dialysis against a 100-0 mM salt gradient for 1 day, then against DI water for 3-4 days before lyophilization.
  • HEPES buffer 10 mM HEPES, 150 mM NaCl, 10 mM EDTA, pH 7.2
  • nanocapsules were synthesized using in situ free-radical polymerization (see, e.g. patent application publication number US-2015-0359752). Briefly, to
  • VEGF vascular endothelial growth factor
  • AAM Acrylamide
  • APM N-(3-aminopropyl)methacrylamide
  • crosslinkers bisacrylated L/D-KNRVK, or methylene bisacrylamide
  • HA hydrogel was formed in 0.3M pH 8.2 HEPES buffer, following steps as below.
  • Tube 1 HA-ADH-Ac in HEPES buffer (ADH modification is 65.62% and Ac modification is 13.33%) was incubated with fibronectin fragments of for 20 min.
  • Tube 2 Polyethylene glycol) dithiol (MW 1000, Sigma-Aldrich, #717142) and Alexa Fluor 555 C2 Maleimide (Thermo Fisher Scientific, #A-20346) solutions in HEPES buffer were mixed together at equal moles for 20 min to generate fresh SH-PEG- AF555. Tube 1 was then mixed with Tube 2 mixture for 20 min before nanocapsules of VEGF was added.
  • MMP Matrix Metallo-protease
  • GMP Matrix Metallo-protease
  • G ratio thiol to acrylate
  • Pre-swelled HA hydrogels 8mm in diameter and 1mm thickness were placed between 8mm (diameter) rheological discs at normal force of 0.01N using a plate-to-plate rheometer (Anton not physica mcr 301 Rheometer). The storage modulus was measured under constant 1% amplitude, from 10 to 0.1 rad/s angular frequency.
  • HA hydrogel was synthesized as described above. Briefly, HA-ADH-Ac is dissolved into 0.08mg/ml solution in 0.3 M HEPES buffer (pH8.2). The solution is then incubated with Fn9* 10 or Fn9(4G)10 for 20 min. SH-PEG-AF555, nanoVEGF, and MMP crosslinker are added sequentially R ratio of 0.60 was used for animal experiment.
  • mice All in vivo studies were conducted in compliance with the NIH Guide for Care and Use of Laboratory Animals and UCLA ARC standards. Seven to nine week old male Balb/c mice were used to study cellular infiltration and blood vessel formation in HA gels with different fibronectin fragments since this strain has been used for wound healing and angiogenesis assay. Mice were anesthetized with 2-3% isoflurane in an induction chamber and kept under anesthesia during the whole surgery. The back of the mouse was shaved, washed with betadine and 70% ethanol. Two lateral incisions appropriate to the size of the implant were made in the skin (one on each side of the midline of the animal) using scissors. Two subcutaneous pockets were subsequently created by blunt dissection using rounded-end scissors.
  • hydrogels were inserted into each respective subcutaneous pocket and closed with a single wound clip. All animals were administered with an anti-inflammatory agent (Carprofen, Rimadyl, 5mg/kg) for the first 48 hours after surgery. At day 7, the clips were taken off. After 2 weeks, each mouse was injected with lOOul of lmg/ml of isolectin GS-IB 4 -AF488 conjugate (ThermoFisher Scientific, #121411) through the left external jugular vein before and sacrificed by isoflurane overdose.
  • an anti-inflammatory agent Carprofen, Rimadyl, 5mg/kg
  • the implant hydrogels (a total of 6 blank gels, 7 Fn9* 10 gels, 7 Fn9(4G)10 gels) were then collected and fixed in 1% PFA for 16 hours at 4°C. Samples were first imaged using a Nikon C2 confocal to visualize the superficial vascular network on the surface of the sample. Light sheet confocal microscopy was then used to image the vascular infiltration in the implanted gel. Briefly, fixed hydrogel samples were inserted into a transparent 6mm tube. The tubes were then filled with 0.3% agarose solution in PBS. After the agarose gel solidified, samples were fixed in position and sheet confocal images were taken at 4x magnification for whole-mount samples (3-5um step size, 6000 images total).
  • HA hydrogel precursor (see Table for composition) was loaded into a 25 ⁇ Hamilton syringe (Hamilton, Reno, NV) connected to a syringe pump. The solution was then injected in liquid form directly into the stroke cavity using a 30-gauge needle at stereotaxic coordinates 0.26 mm anterior/posterior (AP), 3 mm medial/lateral (ML), and 1 mm dorsal/ventral (DV) with an infusion speed of 1 ⁇ /min. The needle was withdrawn from the mouse brain immediately after the injection was complete.
  • Hydrogel composition see Table for composition
  • mice injected with fibronectin fragment Vs+ Fn9* 10, nV+Fn9* 10 and nV+Fn9(4G)10) containing hydrogels were perfused with DyLight 594 labeled Lycopersicon Esculentum (Tomato) Lectin (Vector Laboratories, # DL-1177) through the left through external jugular vein and then sacrificed by isoflurane overdose.
  • Other mice conditions No gel, HA-RGD and Vs+HA-RGD were perfused with 4% PFA and sacrificed.
  • mice brains were harvested and post-fixed in 4% PFA overnight or perfused with PFA before harvesting, then cryoprotected in 30% sucrose in phosphate buffer for 24 hours and frozen. Tangential cortical sections of 30 ⁇ -thick were sliced using a cryostat and directly mounted on gelatin-subbed glass slides. Brain sections were then washed in PBS and permeabilized and blocked in 0.3% Triton and 10% Normal Donkey Serum before being immunohistochemically stained.
  • Rat anti-Ter-119 R&D Systems, #MAB1125, 1 :200
  • Rabbit anti-Glut- 1 Glucose Transporter!, Abeam, 1 :400
  • Donkey anti-rat and rabbit- AF488 Thermo Fisher Scientific, 1 :200
  • the slides were dehydrated in ascending ethanol baths, dewaxed in xylene and coverslipped over fluorescent mounting medium (Dako).
  • the vascular area (stained by Glut-1 only or by both tomato lectin and Glut-1) in the infarct and peri-infarct areas was quantified in 8 randomly chosen regions of interest (ROI) of 0.3 mm 2 in both regions.
  • ROI regions of interest
  • the positive area was measured using pixel threshold on 8-bit converted images (ImageJ vl .43, Bethesda, Maryland, USA) and expressed as the area fraction of positive signal per ROI. Values were then averaged across all ROI and sections, and expressed as the average positive area per animal.
  • perfused vascular ramifications allows for a quantitative analysis of the vessel architecture, by counting manually the number of branching points on positively tomato lectin perfused vessels of the peri-infarct per mm 2 .
  • GLDSPTGIDFSDITANSFTVHWIAPJAATITGYRIRHHPEHFSGRPREDRVPHSRN SITLTNLTPGTEYVVSIVALNGREESPPLIGQQSTVSDVPRDLEVVAATPTSLLI SWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVY AVTGRGDSPASSKPISINYRT (SEQ ID NO: 1) ⁇ - ⁇ 3 Binding Polypeptide GLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRN SITLT LTPGTEYVVSIVALNGREESPPLIGQQSTVSXDVPRDLEVVAATPTSLL ISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITV YAVTGRGDSPASSKPISINYRT wherein X comprises between 2 and 10 heterologous amino acids (SEQ ID NO: 2)
  • VEGF-A Vascular endothelial growth factor 165 (Ala 27-Arg 191)
  • VEGF-A 165 was supplied by Genentech USA.
  • the Sequence from which this is derived is NCBI Reference Sequence: NP_001165097.1 :
  • NQEQVSPL (SEQ ID NO: 6)
  • GCGYGRGDSPG-NQEQVSPL (SEQ ID NO: 7)
  • KNRVK (SEQ ID NO: 8)
  • MMP Matrix Metallo-protease
  • Inserted Factor XHIa crosslinking site (first 8 amino acids from alpha-2 plasmin inhibitor).

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Abstract

La liaison de l'intégrine à des échafaudages d'hydrogel mis au point par génie biologique est essentielle pour la repousse et la régénération tissulaires, mais toute liaison de l'intégrine ne peut donner lieu à une réparation tissulaire. La présente invention a démontré que, par le biais de matériaux d'hydrogel mis au point par génie biologique destinés à favoriser la liaison de l'intégrine α3/α5β1, il est possible de favoriser la formation d'un système vasculaire plein et mature par comparaison avec des matériaux d'hydrogel qui favorisent une liaison de l'intégrine αvβ3 (par exemple RGD). In vivo, les échafaudages α3/α5β1 délivrant un facteur de croissance de l'endothélium vasculaire (FCEV) favorisent des infiltrations de vaisseaux sanguins non sinueux et des vaisseaux sanguins à imperméabilité dans les 10 qui suivent un accident vasculaire cérébral. En revanche, des matériaux qui favorisent la liaison de l'intégrine αvβ3 ont favorisé l'agglutination de bourgeons endothéliaux in vitro et des vaisseaux non imperméables in vivo. La présente invention montre pour la première fois qu'une activation de l'intégrine contrôlée avec précision à partir d'un biomatériau peut être exploitée pour diriger une régénération de vaisseaux thérapeutique et réduire la perméabilité vasculaire induite par FCEV in vivo.
PCT/US2017/051616 2016-09-14 2017-09-14 Matériaux de stimulation de l'intégrine pour la normalisation d'un système vasculaire malade WO2018053166A2 (fr)

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