WO2018053166A2

WO2018053166A2 - Integrin stimulating materials for the normalization of diseased vasculature

Info

Publication number: WO2018053166A2
Application number: PCT/US2017/051616
Authority: WO
Inventors: Tatiana Segura; Thomas H. Barker; Shuoran LI
Original assignee: The Regents Of The University Of California
Priority date: 2016-09-14
Filing date: 2017-09-14
Publication date: 2018-03-22
Also published as: WO2018053166A3

Abstract

Integrin binding to bioengineered hydrogel scaffolds is essential for tissue regrowth and regeneration, yet not all integrin binding can lead to tissue repair. Here we show that through engineering hydrogel materials to promote α3/α5β1 integrin binding, we can promote the formation of a space filling and mature vasculature compared to hydrogel materials that promote a αvβ3 integrin binding (e.g. RGD). In vivo, α3/α5β1 scaffolds delivering vascular endothelial growth factor (VEGF) promoted non-tortuous blood vessel infiltration and non-leaky blood vessels by 10 days post stroke. In contrast, materials that promote αvβ3 integrin binding promoted endothelial sprout clumping in vitro and leaky vessels in vivo. This work shows for the first time that precisely controlled integrin activation from a biomaterial can be harnessed to direct therapeutic vessel regeneration and reduce VEGF induced vascular permeability in vivo.

Description

INTEGRIN STIMULATING MATERIALS FOR THE NORMALIZATION OF

DISEASED VASCULATURE

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under Section 119(e) from U.S. Provisional Application Serial No. 62/394,694, filed September 14, 2016, entitled "INTEGRIN STIMULATING MATERIALS FOR THE NORMALIZATION OF DISEASED VASCULATURE", the contents of which are incorporated herein by reference. STATEMENT AS TO RIGHTS TO INVENTION MADE UNDER

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant Number NS079691, awarded by the National Institutes of Health. The Government has certain rights in the invention.

TECHNICAL FIELD

The invention relates to methods and materials useful for vascular patterning including vascular morphology control and VEGF permeability reduction.

BACKGROUND OF THE INVENTION

The design of 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 (β) 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 β heterodimers (ανβ3, ανβ5, ανβΐ, αΐβΐ, α2β1, α3β1 and α5β1) 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 βΐ and β3 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). Further, both up-regulation and abolishment of βΐ and β3 integrin activation have shown to be related to pathological angiogenesis (see, e.g. 1, 11-16). For example, while excessive suprabasal expression of βΐ integrin in skin has been shown to induce a psoriasis phenotype (see, e.g. 15), the knockout of βΐ integrin resulted in the weakening of endothelial cell junctions and induction of blood leakage in a retinal angiogenesis assay (see, e.g. 11). Likewise, the upregulation β3 integrin leads to enhanced endothelial cell permeability (see, e.g. 17) while the abolition of β3 integrin leads to intrauterine bleeding, defective coronary capillaries, and enhanced tumor angiogenesis (see, e.g. 12, 13, 16). Thus, 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.

The incorporation of integrin binding peptides derived from natural ECM proteins to biomaterials 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. Though 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. However, due to the enormous size and complexity of full-length ECM proteins, the expression process is difficult and an alternative approach to present peptide motifs in a native 3D structural context is needed. One approach is to express only the integrin binding domains from the full-length protein, which is significantly smaller and less complex. For example, recombinant 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). However, without the rest of the full-length protein 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.

Accordingly, there is a need for methods and materials that allow for more precise control over integrin activity in order to, for example, modulate endothelial cell physiology in a way that directs therapeutic vessel regeneration and reduce VEGF induced vascular permeability in vivo.

SUMMARY OF THE INVENTION

The fibronectin 9th type III repeat and 10th type III repeat has been engineered to promote α3β1 and α5β1 ("α3/α5β1") 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. As disclosed in detail below, have discovered that the specific engagement α3/α5β1 or ανβ3 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. For example, we have discovered that certain fibronectin polypeptides can preferentially engage α3/α5β1, and alternatively, certain fibronectin polypeptides can preferentially engage ανβ3 integrin heterodimers. Unexpectedly, the selective engagement of α3/α5β1 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 ανβ3 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 α3/α5β1 or ανβ3 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. In particular, as discussed in detail below, 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. The methods and materials disclosed herein show that 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.

Objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures la-i show that HUVEC sprouting is greatly affected by integrin activation. (Figure la) Representative immunofluorescent images for sprouting in both Fibl and Fib3 fibrin gel. Scale bar: 100 μπι. (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 μιη. (Figure 2b) Intra-loop and intra- joints structures in Fib3 gel with 1000 μΜ RGD. Scale bar: 50 μιη. (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 μΜ RGD Fib3 gels. (Figure 2f) Microscopic analysis of whole bead sprouting effects integrin (scale bar: 100 μπι) and intra-joint and intra-loop branch structures (scale bar: 50 um) from blockage of αν, a5, βΐ or β3 on 2 μΜ Fn9* 10 or 2 μΜ Fn9(4G)10 Fibl gels. (Figure 2g) Branch cluster occurrence comparison between 2 μΜ Fn9(4G)10 Fibl gels with and without av blocking (n=51). * ** *** and **** indicate P < 0.05, P < 0.01, P < 0.001 and P < 0.0001, respectively.

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 μΜ Fn9* 10 and 2 μΜ Fn9(4G)10 Fibl gels. Scale bar: 200 um. (Figures 3b, 3e, and 3h) Sample 1 of bead-bead interactions. Scale bar: 100 μπι. (Figures 3c, 3f, and 3i) Sample 2 of bead-bead interactions. Scale bar: 100 μπι.

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 μπι. ** indicate P < 0.01. (Figure 4d) Microscopic analysis of VE- cadherin signals from blockage of av integrin on 2 μΜ Fn9(4G)10 Fiblgels in comparison to 2 μΜ Fn9* 10 Fibl gels. Scale bar: 100 μπι (whole bead) and 50 μπι (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 μΜ fragment-loaded HA hydrogels. Scale bar: 200 μπι. (Figure 5d) Vessel tortuosity comparison between 9* 10 and 9(4G)10 conditions. Scale bar: 50 μιη. (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 μπι. ** 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. In order to protect the healthy parenchyma from nearby lesion area, star-shaped glial cells, astrocytes, elongate and surround the damaged site, forming the astrocytic scar. 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 μπι. 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. DETAILED DESCRIPTION OF THE INVENTION

In the description of embodiments, reference may be made to the accompanying figures which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention. All publications mentioned herein are incorporated herein by reference to disclose and describe aspects, methods and/or materials in connection with the cited publications (e.g. Li et al., Nat Mater. 2017 Sep; 16(9):953-961).

Many of the techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art. Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

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 ανβ3 integrin heterodimers and α3/α5β1 integrin heterodimers. In this embodiment of the invention, the method comprises selecting a fibronectin polypeptide comprising SEQ ID NO: 1 to preferentially bind o3l α5β1 integrin heterodimers within the population of integrin heterodimers; or selecting a fibronectin polypeptide comprising SEQ ID NO: 2 to preferentially bind ανβ3 integrin heterodimers within the population of integrin heterodimers. In this method, 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 ανβ3 integrin heterodimers or α3/α5β1 integrin heterodimers within the population of integrin heterodimers.

Typically in the invention, the fibronectin polypeptide of SEQ ID NO: 1 or the polypeptide SEQ ID NO: 2 is covalently coupled to a hydrogel composition. Certain embodiments comprise 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.). Optionally the hydrogel is crosslinked by degradable crosslinkers such as protease degradable peptides. In embodiments of the invention, the hydrogel composition further comprises human vascular endothelial growth factor (VEGF) having the amino acid sequence shown in SEQ ID NO: 3. Typically, 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. Optionally the crosslinking agents selected to degrade within an in vivo environment comprise protease degradable peptides formed from D and L amino acids. In certain embodiments of the invention, 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. In the working embodiments of the invention that are disclosed herein, 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. In this embodiment of the invention Fn III9 comprises SEQ ID NO: 4, Fn III10 comprises SEQ ID NO: 5 and the polypeptide preferentially binds α3β1 and/or α5β1 integrin heterodimers. Typically, 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 α3/α5β1 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. Typically in this method, 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. Optionally these 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). In this embodiment, Fn III9 comprises SEQ ID NO: 4, Fn III10 comprises SEQ ID NO: 5, and 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 α5β1 integrin. Certain embodiments comprise avP3-integrin and α5β1 -integrin, with amounts of ανβ3- integrin bound to the polypeptide being greater than amounts of a5pi-integrin or α3β1 -integrin bound to the polypeptide. Certain embodiments further comprise a hydrogel covalently coupled to the polypeptide. Optionally 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. This 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 α3/α5β1 integrin heterodimers as compared to avP3-integrin heterodimers.

Further illustrative aspects and embodiments of the invention are discussed below. Fibronectin fragments with tunable integrin binding

Recombinant fibronectin fragments of the 9th type III repeat (Fn III9) and 10th type III repeat (Fn III10) were designed to preferentially bind α3/α5β1 or ανβ3 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 βΐ integrins, including α5β1 and α3β124, (see, e.g. 29, 41). Four (4) glycine residues were then inserted into the linker region between Fn III9 and Fn III10. The 4xGly insertion both physically separates the synergy (PHSRN) and RGD sites located on Fn III9 and Fn III 10, respectively, and introduces torsional flexibility between the two domains, resulting in a complete disruption of α5β1 integrin binding and promoting a ανβ3 -integrin preference. Though both recombinant fragments can theoretically bind ανβ3 integrin via the RGD sequence, we and others consistently observe a preference of the stabilized mutant to bind synergy-dependent integrins, like α5β1 integrin in cell material interactions (see, e.g. 31, 42). In this disclosure, we call the Leu-Pro mutated, or stabilized, fragment 9* 10 and the 4xGly insertion mutated fragment 9(4G)10. For ease of immobilization onto surfaces and incorporation into natural and synthetic hydrogel biomaterials both fragments were produced with an N- terminal cysteine residue to allow Michael type addition modifications and a factor XHIa substrate sequence, consisting of residues 1-8 of the protein alpha2 plasmin inhibitor (α2ΡΙ1-8, NQEQVSPL, SEQ ID NO: 6) (see, e.g. 43) to allow enzymatic conjugation.

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/cm²) for both fragments, indicating that the reactivity of both fragments is similar. The response of 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 ανβ3, validating that the fragment Fn9* 10 does not mediate significant binding through ανβ3. 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.

Growth factors are critical for the process of angiogenesis and their incorporation within therapeutic angiogenic materials has been a major focus of the field. Here we use vascular endothelial growth factor A 165 (VEGF), which has been described as the master regulator of angiogenesis (see, e.g. 44), to induce EC sprouting and angiogenesis in vivo. Exposure of ECs plated on Fn9* 10 or Fn9(4G)10 did not change the binding to ανβ3, 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

Next, we looked at the influence of fibronectin mediated cell adhesion on vascular endothelial growth factor A 165 (VEGF) induced vascular sprouting. To study the role of fibronectin in EC sprouting, 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. Sprouting and branching points in 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. However, 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.

We next tested the role of full length fibronectin, Fn9* 10 and Fn9(4G)10 containing matrices of EC sprouting. The number of sprouts per bead showed no significant difference among full length fibronectin, Fn9* 10 and Fn9(4G)10 conditions; however, the branch points per bead between full length fibronectin and Fn9(4G)10 conditions is significantly different, indicating that integrin engagement and specificity can affect the branch outcomes even when they induce similar number of sprouts. While no significant difference in sprouting was observed between the two fragment conditions in both Fibl and Fib3 gels (Fig. lb,c&g,h), the addition of 9(4G)10 fibronectin fragment to fibronectin containing matrices (Fibl gels) resulted in a statistical reduction of the number of sprouts per bead compared with blank gel, providing evidence that ανβ3 binding enhancement in fibrin matrices with endogenous fibronectin can affect sprouting patterns (Fig. lg). The fact that the addition of both 9(4G)10 or 9* 10 to fibronectin containing matrices results in increased branching points leads us to investigate the induction of tip cells for matrices modified with the different fragments (Fig. lh).

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. This apparent contradiction lead us to examine the entire sprouting network for other morphological changes to the sprouting vascular pattern. We observed sprouting "clusters" in the Fn9(4G)10 modified matrices but not on blank, Fn, or Fn9* 10 modified matrices (Fig. la). Using high resolution z-stack confocal images we found that the clusters were associated with intra-loop and intra-joint structures both within and between neighboring sprouts which were not observed in Fn9* 10 modified matrices (Fig. 2a). The quantification of the number of branch clusters per beads showed statistically significant occurrence in Fn9(4G)10 gels compared with the blank, native Fn and Fn9* 10 modified Fibl or Fib3 matrices (Fig.2c). These intra-loop and intra-joint features represent chaotic tumor-like vasculature with over-branched and excessive-shunt vessels (see, e.g. 48). Taken together, these results demonstrate that although both Fn fragments enhanced sprouting and branching of ECs in vitro, they lead to different vascular patterns in the resulting EC network. In particular, Fn9(4G)10 promoted the formation of a pathological vascular network containing intra-loop and intra-joint features.

To further confirm the involvement of specific integrin binding in VEGF induced EC sprouting, inhibition of integrin binding through function blocking antibodies was used. In both Fibl or Fib3 gels, blocking of av showed similar levels of EC sprouting as seen without blocking, while blocking with of α5, β3 and βΐ completely prevented the sprouting process at two different Fn9* 10 and Fn9(4G)10 dosages (Fig.2f). These results further illustrate the importance of integrin binding to biomaterials to promote angiogenesis and provides evidence that av is not essential for VEGF induced EC sprouting. We next explored whether the blocking av normalized the resulting vasculature by decreasing the number of with intra-loop and intra-joint structures. Blocking of av significantly decreased the number of branch clusters, further confirming the role of av integrin binding in branch cluster formation (Fig- 2g). Matrices modified with ανβ3 binding peptides show similar vascular pattern as Fn9(4G)10

Next, we sought to confirm the observed vascular pattern in Fn9(4G)10 modified matrices utilizing the ubiquitously used synthetic peptide derived from fibronectin, RGD. RGD has been demonstrated to engage integrins primarily through ανβ3 integrins49 and thus should demonstrate similar vascular pattern as Fn9(4G)10. RGD peptide (GCGYGRGDSPG-NQEQVSPL, SEQ ID NO: 7)) was incorporated within fibrin matrices using the same FXIIIa chemistry used for the incorporation of our fibronectin fragments. As observed with the fibronectin fragments, the incorporation of RGD within Fib3 matrices resulted in enhanced EC sprouting (Fig. 2e). As expected, similar intra-loop and intra-joint structures were found in RGD modified fibrin matrices (Fig. 2b) and the number of clumps were statistically increased for matrices containing ΙΟΟΟμΜ RGD peptide (Fig.2d). These results confirm that ανβ3 integrin binding promotes the formation of a disordered vasculature. It should be noted that the effect of enhanced sprouting and disorganized structures was only observed for a much higher concentration of RGD peptide (> 500μΜ) compared with the concentration of fibronectin fragments used (2μΜ); this result confirms that fibronectin fragments more efficiently display the RGD motif to cells resulting in more efficient binding.

Upregulation of ανβ3 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. We do not mean to suggest that the use of 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 βΐ integrin (see, e.g. 57) and immobilization of VEGF leads to βΐ 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. In healthy vessels, once a vessel branch is formed, the majority of endothelial cells become quiescent, among which only 0.01% still divide (see, e.g. 47). During the angiogenesis process, 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. To test the effect of ανβ3 and α3/α5β1 on EC sprout anastomosis, the same bead assay was performed using stably transfected ECs expressing enhanced green fluorescent protein (EGFP). 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 α3/α5β1 integrin engagement supports similar anastomosis as native fibronectin present in the Fibl matrix. In contrast, ανβ3 integrin engagement through Fn9(4G)10 modified matrices promoted multiple tip-tip contacts, resulting in independent contact sites and loop structures. Thus, consistent with our observations in EC sprouting morphogenesis, inter-loop and inter-joint structures are observed in anastomosed sprouts within Fn9(4G)10 modified matrices (Fig. 3). av activation leads to pathological vasculature through VE-cadherin disruption

Next, we investigated possible mechanisms for the observed differences in the vascular patterns generated by Fn9* 10 and Fn9(4G)10 modified matrices. Failure to generate single tip-tip contact sites during anastomosis with Fn9(4G)10 modified matrices suggested disturbed polarization events between interacting tip cells and disrupted VE-cadherin signaling (see, e.g. 62). VE-cadherin is known to be necessary for the generation of a single polarization event between interacting tip cells (see, e.g. 62). Vessel sprouts lacking VE-cadherin display irregular anastomosis, characterized by multiple tip-tip contact sites and disturbed junctional connections (see, e.g. 62), similar to our observations in Fn9(4G)10 condition. 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 ανβ3 integrin, enhancing endothelial cell permeability (see, e.g. 17). Thus, we hypothesize that ανβ3 activating scaffolds lead to pathological intra- vessel and inter-vessel features through VE-cadherin disruption.

To verify our hypothesis, we first examined VE-cadherin distribution on EC cultured in vitro on surfaces coated with Fn9* 10 or Fn9(4G)10. Cells seeded on Fn9* 10 surface showed significantly increased amount of VE-cadherin signal at cell- cell junctions compared with Fn9(4G)10 condition both without and with VEGF presence (Fig. 4a). Obvious absence and significantly lowered activation of VE- cadherin between adjacent cells were observed on Fn9(4G)10 surfaces, indicating VE-cadherin disruption (Fig.4 a-c). 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). To confirm that increased av integrin binding is responsible for the decrease in VE-cadherin staining, 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). Taken together, these findings support our hypothesis that VE-cadherin-related pathological vasculature was caused by av activation and demonstrate that av blockage can be utilized to rescue the pathological effects.

Integrin stimulation from a bioengineered matrix guides vascular patterns in vivo

The fibronectin fragments were also tested in a modified matrigel plug assay that uses bioengineered hyaluronic acid (HA) hydrogels instead of matrigel to assess angiogenesis in mice. 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. By changing the amino acids in the peptide crosslinker from L enantiomer to D enantiomer, the kinetics of enzymatic cleavage are modulated to control the release rate of VEGF over time. We have previously shown that by mixing fast and slow degrading nanocapsules we can achieve a release rate that can promote vascularization in skin and brain leading to enhanced wound closure (see, e.g. 66, 68). Here we utilized nanocapsules crosslinked by the plasmin-degradable peptide KNRVK (SEQ ID NO: 8). We synthesized four different VEGF nanocapsules containing 100% L, 75% L, 50% L and 25% L crosslinker (Fig. 5a) and mixed them at equal amounts to achieve sustained VEGF release. Hydrogels containing none or 10μΜ 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. 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).

Next, light sheet fluorescent microscopy was used for large-scale whole- mount gel scan (approximately 8mm in diameter, 3mm in thickness) in order to evaluate the penetration distance of vessels. Through the 3D heat map view, we were able to visualize vessel penetration starting from gel surface (Fig. 5e). Projection view was later acquired via dividing whole-scanned 3D gel structure into lOOum thickness slices and performing maximum intensity projection. This helped us visualizing penetration distance of vessels. As expected, both Fn9* 10 and Fn 9(4G)10 gels had extensive vessel infiltration (Fig. 5e,f). Fn9* 10 showed a significantly increased perpendicular maximum infiltration distance when compared with blank gel (Fig.5g). α3/α5β1 integrin binding reduces VEGF induced vascular permeability after stroke

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). However, 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). Thus, effective VEGF delivery is a holy grail in the field of therapeutic angiogenesis. In the brain, VEGF is one of the essential molecules in normal post-stroke angiogenesis (see, e.g. 79); however, the delivery of VEGF after stroke has been complicated by the induction of a disordered and permeable vasculature (see, e.g. 80, 81). Thus, we next sought to evaluate the angiogenic response of integrin stimulation on VEGF-induced angiogenesis in a murine model of brain stroke. Adult 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 10μΜ fibronectin fragments (nV+Fn9(4G)10 and nV+Fn9* 10 directly into the stroke cavity (Fig. 6a). Animal control groups were transplanted with either HA-RGD hydrogel alone (RGD), HA hydrogel containing soluble VEGF and RGD (Vs+RGD) or the star fragment (Vs+ Fn9* 10) or nothing (No gel). Ten days post-transplantation, animals injected with fibronectin fragment containing hydrogels were perfused with tomato lectin before sacrifice for the purpose of studying perfused vessel morphology while other animals were directly perfused with 4% PFA and sacrificed.

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). To compare the vascular bed in all conditions, 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. As expected, all the VEGF containing hydrogels showed a greater vasculature area percentage than RGD only gels in the infarct and the peri-infarct regions.

However, we found that the 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 α3/α5β1 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.

In order to evaluate the quality of these newly formed vascular network, both their permeability and structure were studied. For this, Ter-119, a red blood cell marker, was fluorescently stained and quantified in terms of positive area in the damaged site (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 α3/α5β1 in promoting vascular permeability and stability while reducing blood leakage (Fig. 6e). In addition, the morphoanalysis of tomato lectin-perfused vessels was performed by quantifying the number of vascular ramification growing out of a common vascular tree. We found that the number of ramification per mm2 was significantly greater in the nV+star condition compared with the nV+4G group, indicating that stimulating α3/α5β1 integrin activation promotes the formation of a physiological vascular architecture.

These results demonstrate for the first time that the therapeutic effect of VEGF on post-stroke angiogenesis can be regulated by the specific integrin stimulation, influencing not only the intensity of the vascular growth but also its vascular patterning and quality.

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 α3/α5β1 or ανβ3 integrin activation. We found that α3/α5β1 and ανβ3 integrin-specific materials (including RGD) regulate endothelial cell 3D branching and anastomosis differently in vitro and in vivo. ανβ3 integrin-specific materials (including RGD) lead to pathological tumor-like sprouting clumps, which can later be rescued by the blockage of av integrin. Our results indicate that the VE-cadherin disruption induced by ανβ3 integrin could be responsible for the development of such pathological feature. Most importantly, α3/α5β1 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.

Immobilization of Fn9* 10 or Fn9(4G)10 on homogenous gold surface

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). A total of lOOmg of EMCH (N-[e-Maleimidocaproic acid]hydrazide, Fisher Scientific, PI-22106) was first dissolved in anhydrous DMSO(Dimethyl sulfoxide) to make 50Mm stock. 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. Mole ratio of COOH in heparin/ EMCH/ NHS/EDC=1 : 1 : 1 : 10. The reaction continued for 6 hours with gentle shaking followed by dialysis. The dialyzed samples were then lyophilized and sent for NMR for modification verification. The modified heparin was then conjugated to SAMs (99% EG-OH, 1% EG-NH2) formed on gold slides via EDC/NHS method as previously stated. Either Fn9* 10 or Fn9(4G)10 (lOOOng/slide, 53.3ng/ cm²) was incubated with the modified heparin- coated surfaces overnight at 4 °C, followed by three PBS washes.

ELISA on gold surface

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. After 3 washes, 60ul/well of TMB substrate (#7004L, Cell signaling) was added, incubated for 8 min in dark and then transferred to 96-well plates containing 1M H2SO4. The absorbance was measured at 450nm and normalized against absorbance at 550nm.

Cell Proliferation Assay

Slides immobilized with Fn9* 10 or Fn9(4G)10 were washed twice with sterile

PBS and then blew dry in cell culture hood for well assembly with 8-wells ibidi sticky-bottom device (ibidi, #80828). A total of 5000 HUVECs in EGM-2 stripped off fibronectin were seeded in each well with or without 2ng/ml of VEGF and the cell number was assayed after 48 hours using Cyquant assay. A minimum of n=8 per condition was used in this experiment.

Cell Migration Assay

Slides were then washed twice with sterile PBS and then blew dry in cell culture hood for well assembly with PDMS sheets containing two 8mm holes. 5000 HUVECs pre-stained with SP-DilC18(3) lipophilic red fluorescence dye (Life Technologies) were seeded in each PDMS well on surface in EGM-2 w/o Fibronectin/VEGF medium and allowed for cell attachment for 3 hours in cell incubator. The slides were then transferred into incubation system of Zeiss LSM 780 confocal for lOx phase time-lapse tracking. Images were taken at 4 different locations over time span of 7 hours with 15 min interval. A MATLAB program was then developed to track the strongest lipid signal. Basically, after circling out manually the target cells, the program tracks the brightest lipid stain in the area to create the cell migration path and calculated cell migration distance. A minimum of n=64 in each condition was used to quantify.

2D Immunofluorescence Staining

Slides immobilized with Fn9* 10 or Fn9(4G)10 were washed twice with sterile

PBS and then blew dry in cell culture hood for well assembly with 12-well customized white Teflon wells. A total of 5000 HUVECs in EGM-2 stripped off fibronectin were seeded in each well with or without 2ng/ml of VEGF and fixed after 24 hrs. Cell samples were first fixed in 4% PFA for 15 min, washed twice with PBS for 5 min each before incubating with PBS+0.1% Triton for 3 min. After washing the samples again with PBS, samples were incubated at room temperature for 30min in blocking buffer: PBS+ 2% Normal Goat Serum. 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 2μg/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.

Sprouting Assay with blank, full length Fibronectin, Fn9* 10 or Fn9(4G)10 fibrin gels 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. The following day, 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),

1 U /ml factor XIII and 0.04 U/ml aprotinin at a pH of 7.4 with or without 2 μΜ Fn9* 10, 2 μΜ Fn9(4G)10, 0.54 μΜ full length Fibronectin (Millipore, FCOlO). A total of 250 ul of this fibrinogen/bead solution was added to 0.16 units of thrombin in one well of glass-bottom 24-well plates. Fibrinogen/bead 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. A total of 20,000 HDFs were plated on top of the clot and the medium was changed every other day. Bead assays were monitored for 7 days. A minimum of n=15 was used for each condition.

Sprouting Assay with RGD Presence

Sprouting assay was performed as previously described in Fib3 fibrin gels with 200, 500 or 1000 μΜ of a₂PIi-8-RGD (H-NQEQVSPLRGDSPG- H2, SEQ ID NO: 9, GenScript).

Anastomosis Sprouting Assay with Fn9* 10 and Fn9(4G)10

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-

2 at 37°C and 5% C02. The following day, beads with cells were 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 Fib3 fibrinogen, 1 U /ml factor XIII and 0.04 U/ml aprotinin, 80,000 cells/ml HDF at a pH of 7.4 with 2 μΜ of Fn9* 10 or Fn9(4G)10. 250 ul of this fibrinogen/bead solution was added to 0.16 units of thrombin in one well of glass- bottom 24-well plates. 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.

Integrin Blocking Sprouting Assay

Sprouting assays were performed as previously described.

For Fn9* 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 μΜ (high dosage) or 0.267 μΜ (low dosage) Fn9* 10 at a pH of 7.4 with or without 5μg/ml of βΐ 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.

For 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 μΜ (high dosage) or 0.239 μΜ (low dosage) Fn9(4G)10 at a pH of 7.4 with or without 5μg/ml of β3 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.

VE-cadherin Staining on 3D Integrin-Blocking Assay

Gel samples were first fixed in 1% PFA for 15 min, blocked for 2 hours at room temperature in a blocking buffer of PBS+ 0.05% Tween-20 + 5% Normal Goat Serum. Samples were then incubated in a primary antibody directed against VE Cadherin (Rabbit, Abeam; ab33168, 1 :200) overnight at 4°C, followed by a secondary antibody (1 :500) and 2μg/ml DAPI for 1 hour. Imaging was performed using a Nikon C2 confocal and images were analyzed using Image J.

VE-cadherin Staining on 2D Fragments-Coated Surfaces

A total of 500ul of 2 μΜ Fn9* 10 or Fn9(4G)10 in PBS buffer was added into sterile 24 well glass-bottom plates (MatTek Corporation) for 1 hour in cell incubator, followed by 0.1% heat-deactivated sterile BSA-PBS buffer for 1 hour. HUVECs cells pre-starved for 6 hours in EBM-2 were collected, resuspended in EGM-2 medium w/o Fibronectin either with or without VEGF and seeded into fragments and BSA treated wells. Cell density of 50,000 cells per well in 24-well glass-bottom plates was used. After 12 hours, cells were fixed in 1% PFA for 15 min and stained for VE-cadherin. Cell samples were first fixed in 1% PFA for 15 min, washed three times with 1XPBS for 5 min before blocked for 2 hrs at room temperature in blocking buffer: 1XPBS+ 0.1%BSA + 0.1% Tween-20 + 0.3M Glycine+ 10% Normal Goat Serum. Samples were incubated in a primary antibody Rabbit anti- VE Cadherin (Abeam; ab3316, 1 :500) overnight at 4°C, followed by a secondary antibodies Donkey anti -Rabbit (1 :500) and 2μg/ml DAPI for 1 hour. Imaging was performed using a Nikon C2 confocal and images were analyzed using Image J.

Hyaluronic Acid-Acrylate Synthesis

Sodium 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. Approximately 60% of the carboxyl groups were modified with ADH, which was determined using ¹H-NMR (D2O) by taking the ratio of peaks at 5 = 1.6 and 2.3 corresponding to the eight hydrogens of the methylene groups on the ADH to the singlet peak of the acetyl methyl protons in HA (δ = 1.88). 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. The degree of acrylation was determined to be ~10% using ¹H-NMR (D2O) by taking the ratio of the multiplet peak at δ = 6.2 corresponding to the cis- and tram'-acrylate hydrogens to the singlet peak of the acetyl methyl protons in HA (δ = 1.88). Vascular Endothelial Growth Factor Nanocapsules Synthesis

The nanocapsules were synthesized using in situ free-radical polymerization (see, e.g. patent application publication number US-2015-0359752). Briefly, to

-3 synthesize n(VEGF), VEGF was diluted in a buffer solution of 10 x 10 M sodium bicarbonate (pH = 8.55) at a final reaction concentration of 100 μg ml \ Acrylamide (AAM) and N-(3-aminopropyl)methacrylamide (APM) and crosslinkers (bisacrylated L/D-KNRVK, or methylene bisacrylamide) were subsequently added to the protein solution (at the molar ratio of VEGF : AAM : APM: crosslinker = 1 :3000:3000:600). Different ratio of L/D crosslinkers were used to generated 100%, 75%, 50% and 25% n(VEGF). Later, these n(VEGF)s were mixed at equal amount for controlled release of VEGF.

HA Hydrogel Storage Modulus Optimization

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. Di-cysteine modified Matrix Metallo-protease (MMP) (Ac- GCRDGPQGIWGQDRCG-NH2, SEQ ID NO: 10) (GenScript) sensitive crosslinker was added in the end to initiate gelation. Gelation was allowed for 30 min at 37°C. To determine the storage modulus range, gels with different thiol to acrylate (R ratio) were tested. 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 paar physica mcr 301 Rheometer). The storage modulus was measured under constant 1% amplitude, from 10 to 0.1 rad/s angular frequency. HA Gel Formula for SubQ Mice Model

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.

SubQ Mice Model and Quantification

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. The 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-IB₄-AF488 conjugate (ThermoFisher Scientific, #121411) through the left external jugular vein before and sacrificed by isoflurane overdose. 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). After 3D rendering, lOOum-thick samples were sliced out to merge into single-plane maximum intensity projection image (none-overlapping samples, 6 different slices from each gel sample). A Matlab program was then developed to detect the maximum infiltration distance of AF555 fluorescently labeled vessels. A minimum of n=35 was used to quantify the maximum infiltration distance in this experiment.

Ischemic Stroke Model

Animal procedures were performed in accordance with the US National

Institutes of Health Animal Protection Guidelines and the University of California Los Angeles Chancellor's Animal Research Committee. Focal and permanent cortical stroke was induced by a middle cerebral artery occlusion (MCAo) on young adult C57BL/6 male mice (8-12 weeks) obtained from Jackson Laboratories. Briefly, under isoflurane anesthesia (2-2.5% in a 70% N2O/30% 02 mixture), a small craniotomy was performed over the left parietal cortex. One anterior branch of the distal middle cerebral artery was then exposed, electrocoagulated and cut. Body temperature was maintained at 36.9 ± 0.4 °C with a heating pad throughout the operation. In this model, ischemic cellular damage is localized to somatosensory and motor cortex.

Brain Hydrogel Transplantation

Five days later, HA hydrogel precursor (see Table for composition) was loaded into a 25 μΐ 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 μΐ/min. The needle was withdrawn from the mouse brain immediately after the injection was complete. Hydrogel composition

Ten days following the hydrogel transplantation, animals 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.

Brain tissue processing

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 μπι -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. The primary antibody Rat anti-Ter-119 (R&D Systems, #MAB1125, 1 :200) or Rabbit anti-Glut- 1 (Glucose Transporter!, Abeam, 1 :400) were incubated overnight at +4°C followed by secondary antibodies Donkey anti-rat and rabbit- AF488 (Thermo Fisher Scientific, 1 :200) for 1 hour at room temperature. After 3x 10 minute washes in PBS, the slides were dehydrated in ascending ethanol baths, dewaxed in xylene and coverslipped over fluorescent mounting medium (Dako).

Microscopy and Morphoanalysis

Analyses were performed on microscope images of 3 coronal brain levels at +0.80 mm, -0.80 mm and -1.20 mm according to bregma, which consistently contained the cortical infarct area. Each image represents a maximum intensity projection of 10 to 12 Z-stacks, 0.85μπι apart, captured at a 20x magnification with a Nikon C2 confocal microscope using the NIS Element software. To quantify the vascular bed in the no gel and gel conditions, 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 lectin-perfused animals the combination of both stains reveals the vascular bed the same as Glut-1 alone in tomato lectin-unperfused animals.

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 mm² in both regions. In each ROI, 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.

The evaluation of 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 mm².

Statistical Analysis

Statistical analyses were performed using Prism (GraphPad, San Diego, CA). Data were analyzed using a one-way analysis of variance (ANOVA) followed by a Tukey post-hoc test. For 2D VE-cadherin staining analysis (n=3) and stroke model quantification (minimum n=5), two-tailed unpaired test was used. The results are expressed as mean ± SEM. Single, double, triple and quadruple asterisks represent p < 0.05, p < 0.01, p < 0.001 and p < 0.0001, respectively. A p value < 0.05 was considered statistically significant. REFERENCES

Note: This application references a number of different publications as indicated throughout the specification as reference numbers. A list of these different publications ordered according to these reference numbers can be found below.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. Publications cited herein are cited for their disclosure prior to the filing date of the present application. Nothing here is to be construed as an admission that the inventors are not entitled to antedate the publications by virtue of an earlier priority date or prior date of invention. Further, the actual publication dates may be different from those shown and require independent verification. NUMBERED REFERENCES CITED IN SPECIFICATION

1. Hynes, R.O. Cell 110, 673-687 (2002).

2. Hynes, R.O. Science 326, 1216-1219 (2009).

3. Giancotti et al., Science 285, 1028-1032 (1999).

4. Hynes et al., Thromb Haemostasis 78, 83-87 (1997).

5. Rupp et al., Circulation research 89, 566-572 (2001).

6. Stupack et al., Sci STKE 2002, pe7 (2002).

7. Zovein, A.C. et al., Dev Cell 18, 39-51 (2010).

8. Bayless et al., Am J Pathol 156, 1673-1683 (2000).

9. Mahabeleshwar, G.H. et al., Cell Cycle 7, 335-347 (2008).

10. Turlo, K.A. et al., Dev Biol 365, 23-35 (2012).

11. Yamamoto, H. et al., Nature communications 6, 6429 (2015).

12. Hodivala-Dilke, K M. et al., J Clin Invest 103, 229-238 (1999).

13. Weis, S.M. et al., Cooperation between VEGF and beta3 integrin during cardiac vascular development. Blood 109, 1962-1970 (2007).

14. Kumar et al., Cancer Metastasis Rev 28, 113-127 (2009).

15. Carroll et al., Cell 83, 957-968 (1995).

16. Reynolds, L.E. et al., Nat Med 8, 27-34 (2002).

17. Alghisi et al., PloS one 4, e4449 (2009).

18. Benoit et al., Biomaterials 26, 5209-5220 (2005).

19. Mhanna, R. et al., Tissue Eng Part A 20, 1165-1174 (2014).

20. Shekaran, A. et al., Biomaterials 35, 5453-5461 (2014).

21. Lee, S.T. et al., Biomaterials 31, 1219-1226 (2010).

22. Gonzalez et al., Tissue Eng 10, 1775-1786 (2004).

23. Mann et al., Biomaterials 22, 3045-3051 (2001).

24. Martino, M.M. et al., Biomaterials 30, 1089-1097 (2009).

25. van der Walle et al., Protein Eng 15, 1021-1024 (2002).

26. Baron, M. et al., Biochemistry 31, 2068-2073 (1992). 27. Krammer et al., Matrix Biol 21, 139-147 (2002).

28. Grant et al., J Biol Chem 272, 6159-6166 (1997).

29. Altroff, H. et al., J Biol Chem 279, 55995-56003 (2004).

30. Martino, M.M. et al., Sci Transl Med 3 (201 1).

31. Brown et al., Tissue Eng Part A 17, 139-150 (2011).

32. Kisiel, M. et al., Biomaterials 34, 704-712 (2013).

33. Martino et al., Faseb J 24, 4711-4721 (2010).

34. Traub, S. et al., Biomaterials 34, 5958-5968 (2013).

35. Wijelath, E. et al., J Biomed Mater Res A 95, 641-648 (2010).

36. Najjar, M. et al., Biotechnol Bioeng 112, 1916-1926 (2015).

37. Brady, A.C. et al., Tissue Eng Part A 19, 2544-2552 (2013).

38. Lin et al., Polymer edition 3, 217-227 (1992).

39. Ghosh et al., Tissue Eng 12, 601-613 (2006).

40. Hou, S. et al., J Neurosci Methods 148, 60-70 (2005).

41. Brown et al., J Biol Chem 290, 25534-25547 (2015).

42. Markowski et al., J Biomed Mater Res A 100, 2119-2127 (2012).

43. Schense et al., Bioconjug Chem 10, 75-81 (1999).

44. Hoeben, A. et al., Pharmacol Rev 56, 549-580 (2004).

45. Nakatsu et al., J Vis Exp, 186 (2007).

46. Nakatsu, M.N. et al., Microvasc Res 66, 102-1 12 (2003).

47. De Smet, F. et al., Arterioscl Throm Vas 29, 639-649 (2009).

48. Carmeliet et al., Nature 407, 249-257 (2000).

49. Mogford et al., Circulation research 79, 821-826 (1996).

50. Voura et al., Molecular biology of the cell 12, 2699-2710 (2001).

51. Friedlander, M. et al., Proc Natl Acad Sci U S A 93, 9764-9769 (1996).

52. Desgrosellier et al., Nat Rev Cancer 10, 9-22 (2010).

53. Henderson et al., Nat Med 19, 1617-1624 (2013).

54. Fiore et al., J Cell Biol 21 1, 173-190 (2015).

55. Liu et al., Drug Dev Res 69, 329-339 (2008).

56. Danhier et al., Mol Pharm 9, 2961-2973 (2012).

57. Lam et al., Biomaterials 34, 3938-3947 (2013).

58. Chen, T.T. et al., J Cell Biol 188, 595-609 (2010). 59. Adams et al., Nat Rev Mol Cell Bio 8, 464-478 (2007).

60. Isogai et al., Development 130, 5281-5290 (2003).

61. Wacker et al., Current opinion in cell biology 23, 676-685 (2011).

62. Lenard, A. et al., Dev Cell 25, 492-506 (2013).

63. Wallez et al., Trends Cardiovasc Med 16, 55-59 (2006).

64. Montero-Balaguer, M. et al., PloS one 4, e5772 (2009).

65. Lei et al., Biomaterials 30, 254-265 (2009).

66. Zhu et al., Adv Mater 27, 3620-3625 (2015).

67. Wen, J. et al., Advanced Materials 23, 4549-4553 (2011).

68. Zhu et al., Ann Biomed Eng 44, 1983-1992 (2016).

69. Yang, J.P. et al., J Invest Surg 23, 149-155 (2010).

70. Taimeh et al., Nat Rev Cardiol 10, 519-530 (2013).

71. Detmar, M. J Dermatol Sci 24 Suppl 1, S78-84 (2000).

72. Alon, T. et al., Nat Med 1, 1024-1028 (1995).

73. Henry, T.D. et al., Circulation 107, 1359-1365 (2003).

74. Kastrup, J. et al., J Am Coll Cardiol 45, 982-988 (2005).

75. Stewart, D.J. et al., Gene Ther 13, 1503-151 1 (2006).

76. Hedman, M. et al., Circulation 107, 2677-2683 (2003).

77. Roberts et al., Journal of cell science 108 ( Pt 6), 2369-2379 (1995).

78. Esser, S. et al., J Cell Biol 140, 947-959 (1998).

79. Andres et al., Stroke (2011).

80. Arai et al., Febs J 276, 4644-4652 (2009).

81. Xiong et al., Curr Opin Investig Drugs 1 1, 298-308 (2010).

POLYPEPTIDE AND POLYNUCLEOTIDE SEQUENCES

il α5-β1 Binding Polypeptide

GLDSPTGIDFSDITANSFTVHWIAPJAATITGYRIRHHPEHFSGRPREDRVPHSRN SITLTNLTPGTEYVVSIVALNGREESPPLIGQQSTVSDVPRDLEVVAATPTSLLI SWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVY AVTGRGDSPASSKPISINYRT (SEQ ID NO: 1) αν-β3 Binding Polypeptide GLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRN SITLT LTPGTEYVVSIVALNGREESPPLIGQQSTVSXDVPRDLEVVAATPTSLL ISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITV YAVTGRGDSPASSKPISINYRT wherein X comprises between 2 and 10 heterologous amino acids (SEQ ID NO: 2)

Vascular endothelial growth factor (VEGF-A) 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 :

MNFLL S W VHW SL ALLL YLHHAKW S Q A APM AEGGGQNHHE VVKFMD V YQR SYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITM QIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQ DPQTCKC SCKNTD SRCK ARQLELNERTCRCDKPRR FIII9 Domain

GLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRN SITLTNLTPGTEYVVSIVALNGREESPPLIGQQSTVS (SEQ ID NO: 4)

FIII10 Domain

DVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKS TATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT (SEQ ID NO: 5) protein alpha2 plasmin inhibitor Site

NQEQVSPL (SEQ ID NO: 6)

RGD peptide

GCGYGRGDSPG-NQEQVSPL (SEQ ID NO: 7)

plasmin-degradable peptide

KNRVK (SEQ ID NO: 8)

RGD Peptide

ΡΙι-8-RGDNQEQVSPLRGDSPG (SEQ ID NO: 9)

cysteine modified Matrix Metallo-protease (MMP)

GCRDGPQGIWGQDRCG (SEQ ID NO: 10)

FnIII9*10 (L to P mutation) NT Sequence: atgggatcttgtaatcaagaacaagtcagtccccttggcttagattctccgactggaattgacttctcagacattacggccaatt ccttcacagtgcactggatcgcaccccgcgcaaccattactggataccgtattcgtcatcaccctgaacacttttcaggacgt ccccgcgaggaccgcgtaccacattcgcgcaacagtatcactcttactaatttgacccctggtactgagtatgtagtttccatc gtcgctctgaacgggcgcgaggaatccccaccgttaattggtcaacaatctaccgtttcagatgttccccgtgatttagaagtt gtagcagctactccgacatctttactgatttcttgggacgcaccagctgtcacagttcgctattaccgcatcacatacggtgaa accggtgggaactcgcctgttcaggaatttactgtgccaggtagtaagtcgaccgcaacaatctccggcttaaagccgggc gtggattatacaattactgtctatgcagttaccggccgcggcgattccccggcgtcgtcaaagccgatcagtatcaattatcg caccggtcaacaatctaccgtttcagatgttccccgtgatttagaagttgtagcagctactccgacatctttactgatttcttggg acgcaccagctgtcacagttcgctattaccgcatcacatacggtgaaaccggtgggaactcgcctgttcaggaatttactgt gccaggtagtaagtcgaccgcaacaatctccggcttaaagccgggcgtggattatacaattactgtctatgcagttaccggc cgcggcgattccccggcgtcgtcaaagccgatcagtatcaattatcgcaccggtggtggagatcatccgccgaaatcagat ttagtgccgcgtggctcgggccatggaacgggctccacgggaagtggtagctcaggtacggcctcgtcggaagataata acatggccgttattaaagaatttatgcgttttaaagttcgtatggaaggttctatgaatggtcatgaatttgaaattgaaggtgaa ggtgaaggtcgtccatatgaaggtactcaaactgctaaattaaaagttactaaaggtggtccattaccatttgcttgggatatttt atctccacaatttatgtatggttctaaagcttatgttaaacatccagctgatattccagactacaagaaactgtcttttccagaag gttttaaatgggaacgtgttatgaattttgaagatggtggtttagttactgttactcaagattcttctttacaagatggtactctgatt tacaaagtcaagatgcgtggtactaattttccaccagatggtccagttatgcaaaaaaaaactatgggttgggaagcttctact gaacgtttatatccacgtgatggtgttttaaaaggtgaaattcatcaagctttaaagctgaaagacggtggccattacctggttg aatttaagacgatctatatggctaagaaaccagttcagctgccaggttactattacgttgatactaagttagatatcacttctcat aacgaagattatactattgttgaacaatatgaacgttctgaaggccgtcatcatttattcttatatggaatggatgagttgtataa gggacacggggggcaccaccaccaccaccatcatcaccatcactga (SEQ ID NO: 11)

FnIII9*10 (L to P mutation) PP Sequence:

MGS(C)¹NQEQVSPLGLDSPTGIDFSDITANSFTVHWIAPRATITGYPJRHHPEHF SGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPi ^LIGQQSTVSDV PRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTA TISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTPPKSD(LVPRGS)²GHGTG STGSGS SGTAS SEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRP YEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFK WERVMNFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMG WEASTERLYPRDGVLKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYY YVDTKLDITSHNEDYTIVEQYERSEGRHHLFLYGMDELYKGHGGHHHHHHH HHH (SEQ ID NO: 12)

FnIII9-4G-10 NT Sequence:

atgggatcttgtaatcaagaacaagtcagtccccttggcttagattctccgactggaattgacttctcagacattacggccaatt ccttcacagtgcactggatcgcaccccgcgcaaccattactggataccgtattcgtcatcaccctgaacacttttcaggacgt ccccgcgaggaccgcgtaccacattcgcgcaacagtatcactcttactaatttgacccctggtactgagtatgtagtttccatc gtcgctctgaacgggcgcgaggaatccccaccgttaattggtcaacaatctaccgtttcaggcggaggtggcgatgttccc cgtgatttagaagttgtagcagctactccgacatctttactgatttcttgggacgcaccagctgtcacagttcgctattaccgca tcacatacggtgaaaccggtgggaactcgcctgttcaggaatttactgtgccaggtagtaagtcgaccgcaacaatctccg gcttaaagccgggcgtggattatacaattactgtctatgcagttaccggccgcggcgattccccggcgtcgtcaaagccgat cagtatcaattatcgcaccggtcaacaatctaccgtttcaggaggaggaggagatgttccccgtgatttagaagttgtagcag ctactccgacatctttactgatttcttgggacgcaccagctgtcacagttcgctattaccgcatcacatacggtgaaaccggtg ggaactcgcctgttcaggaatttactgtgccaggtagtaagtcgaccgcaacaatctccggcttaaagccgggcgtggatta tacaattactgtctatgcagttaccggccgcggcgattccccggcgtcgtcaaagccgatcagtatcaattatcgcaccggt ggtggagatcatccgccgaaatcagatttagtgccgcgtggctcgggccatggaacgggctccacgggaagtggtagct caggtacggcctcgtcggaagataataacatggccgttattaaagaatttatgcgttttaaagttcgtatggaaggttctatgaa tggtcatgaatttgaaattgaaggtgaaggtgaaggtcgtccatatgaaggtactcaaactgctaaattaaaagttactaaag gtggtccattaccatttgcttgggatattttatctccacaatttatgtatggttctaaagcttatgttaaacatccagctgatattcca gactacaagaaactgtcttttccagaaggttttaaatgggaacgtgttatgaattttgaagatggtggtttagttactgttactcaa gattcttctttacaagatggtactctgatttacaaagtcaagatgcgtggtactaattttccaccagatggtccagttatgcaaaa aaaaactatgggttgggaagcttctactgaacgtttatatccacgtgatggtgttttaaaaggtgaaattcatcaagctttaaag ctgaaagacggtggccattacctggttgaatttaagacgatctatatggctaagaaaccagttcagctgccaggttactattac gttgatactaagttagatatcacttctcataacgaagattatactattgttgaacaatatgaacgttctgaaggccgtcatcattta ttcttatatggaatggatgagttgtataagggacacggggggcaccaccaccaccaccatcatcaccatcactga (SEQ ID NO: 13)

FnIII9-4G-10 PP Sequence:

MGS(C)¹NQEQVSPLGLDSPTGIDFSDITANSFTVHWIAPRATITGYPJRHHPEHF SGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPi ^LIGQQSTVSiG GGG) ³D VPRDLE V V A ATPT SLLI S WD AP A VT VRY YRIT YGETGGNSP VQEF TV PGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTPPKSDiLVPRG S£GHGTGSTGSGS SGTAS SEDN MAVIKEFMRFKVRMEGSMNGHEFEIEGEG EGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKL SFPEGFKWERVMNFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVM QKKTMGWEASTERLYPRDGVLKGEIHQALKLKDGGHYLVEFKTIYMAKKPV QLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRHHLFLYGMDELYKGHGGH HHHHHHHHH (SEQ ID NO: 14)

(Footnote) and Other Sequence Legends:

1. Inserted Cys for thiol-conjugation.

2. Inserted Factor XHIa crosslinking site (first 8 amino acids from alpha-2 plasmin inhibitor).

3. Mutations (P is the Leu to Pro mutation; 9* 10 and GGGG is 4-Gly insertion).

6. NQEQVSPL- thrombin seq.

7.

VIKEFMRFKVRMEGSMNGHEFEIEGEGEGRP YEGTQT AKLKVTKGGP

LPF AWDIL SPQFM YGSK AYVKUP ADIPD YKKL SFPEGFKWERVMNFEDGGL V TVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDY TIVEQYERSEGRHHLFL- tdtomato fusion.

8. HHHHHHHHHH 10 His residues (for purification).

9. Underlined - final protein product used in studies.

CONCLUSION

This concludes the description of the preferred embodiment of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching.

Claims

CLAIMS:

1. A method of using a fibronectin polypeptide to preferentially bind one or more integrin heterodimers within a population of integrin heterodimers that includes ανβ3 integrin heterodimers and α3/α5β1 integrin heterodimers, the method comprising:

(a) selecting a fibronectin polypeptide comprising SEQ ID NO: 1 to preferentially bind ο3Ι α5β1 integrin heterodimers within the population of integrin heterodimers; or

(b) selecting a fibronectin polypeptide comprising SEQ ID NO: 2 to preferentially bind ανβ3 integrin heterodimers within the population of integrin heterodimers;

(c) combining a polypeptide of (a) or (b) with the population of integrin heterodimers; and

(d) allowing the fibronectin polypeptide of (a) or (b) to preferentially bind ανβ3 integrin heterodimers or α3/α5β1 integrin heterodimers within the population of integrin heterodimers;

so that αν-β3 integrin heterodimers or α3/α5β1 integrin heterodimers within the population of integrin heterodimers are preferentially bound.

2. The method of claim 1, wherein the fibronectin polypeptide of (a) or (b) is covalently coupled to a hydrogel composition.

3. The method of claim 2, wherein the hydrogel is crosslinked by protease degradable peptides.

4. The method of claim 3, wherein the hydrogel composition further comprises human vascular endothelial growth factor (VEGF) having the amino acid sequence shown in SEQ ID NO: 3.

5. The method of claim 4, wherein 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.

6. The method of claim 5, wherein the crosslinking agents selected to degrade within an in vivo environment comprise protease degradable peptides formed from D and L amino acids

7. The method of claim 1, wherein the fibronectin polypeptide comprises SEQ ID NO: 1 coupled to a heterologous amino acid sequence.

8. The method of claim 7, wherein 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.

9. The method of claim 8, wherein binding of the fibronectin polypeptide to the vascular endothelial cell modulates at least one of:

endothelial cell vessel sprouting;

filopodia development in endothelial tip cells.

10. A method of modulating vessel sprouting in human endothelial cells comprising:

(a) combining the endothelial cells with a composition comprising:

a 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 III 10); and

human vascular endothelial growth factor (VEGF) comprising the amino acids in SEQ ID NO: 3;

wherein:

Fn III9 comprises SEQ ID NO: 4;

Fn III 10 comprises SEQ ID NO: 5; and the polypeptide preferentially binds α5β1 integrin heterodimers; and (b) allowing the polypeptide to bind to α3/α5β1 integrin heterodimers expressed by the endothelial cells and allowing the VEGF to bind to VEGF receptors on the endothelial cells so that vessel sprouting in the endothelial cells is modulated.

11. The method of claim 10, wherein 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, wherein said peptides comprise selected amounts of D and L amino acids.

12. The method of claim 11, wherein the fibronectin polypeptide is coupled to a heterologous amino acid sequence comprising a protease recognition site.

13. The method of claim 12, wherein the hydrogel comprises hyaluronic acid moieties.

14. The method of claim 13, wherein the endothelial cells are disposed in a wound.

15. A composition of matter comprising:

a polypeptide comprising a 9th type III repeat of fibronectin (Fn III9) and a 10th type III repeat of fibronectin (Fn III 10);

wherein:

Fn III9 comprises SEQ ID NO: 4;

Fn III 10 comprises SEQ ID NO: 5;

Fn III9 and Fn III 10 are linked together by a heterologous amino acid linker comprising at least two amino acid residues; and

the polypeptide preferentially binds avP3-integrin as compared to α5β1 integrin.

16. The composition of claim 15, further comprising avP3-integrin and α5β1- integrin, wherein amounts of ανβ3 -integrin bound to the polypeptide are greater than amounts of α5β1 -integrin bound to the polypeptide.

17. The composition of claim 16, wherein Fn III9 and Fn III10 are linked together by an amino acid linker comprising two to five amino acid residues.

18. The composition of claim 17, wherein Fn III9 and Fn III 10 are linked together by an amino acid linker comprising at least two glycine residues.

19. The composition of claim 18, further comprising a hydrogel, wherein:

the hydrogel is covalently coupled to the polypeptide; and the hydrogel is crosslinked by protease degradable peptides.

20. 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;

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 III10);

wherein:

Fn III9 comprises SEQ ID NO: 4;

Fn III 10 comprises SEQ ID NO: 5; and

the polypeptide preferentially binds α3/α5β1 integrin heterodimers as compared to ανβ3 -integrin heterodimers.