WO2009004544A2

WO2009004544A2 - Artificial vessels, kits and methods

Info

Publication number: WO2009004544A2
Application number: PCT/IB2008/052578
Authority: WO
Inventors: Han Li
Original assignee: Tian, Ye; Xia, Han
Priority date: 2007-06-29
Filing date: 2008-06-26
Publication date: 2009-01-08
Also published as: WO2009004544A3

Abstract

An artificial vessel comprises elastic protein and vessel growth stimulator capable of stimulating vessel endothelial cell growth. Another artificial vessel comprises a tube formed of elastic protein and at least one element attached to the tube for physically stimulating vessel endothelial cell growth. Implant kits comprise such artificial vessels. Methods of manufacturing an artificial vessel using recombinant techniques comprise inducing expression of an elastic protein- vessel growth stimulator fusion protein from a fusion gene of an elastic protein and a vessel growth stimulator.

Description

ARTIFICIAL VESSELS, KITS AND METHODS

FIELD OF THE INVENTION

[0001] The present invention relates to artificial vessels which may be used as implants. The vessels comprise a combination of elastic protein, an example of which is resilin, and vessel growth stimulator, an example of which is angiogenin. The present invention further relates to kits including such artificial vessels and methods for production of such artificial vessels.

BACKGROUND OF THE INVENTION

[0002] The human body has a network of blood vessels circulating oxygen, nutrients and other molecules that are essential for life. Vessel abnormalities can lead to severe problems which may threaten life or devastate the quality of life for an individual. For example, in a traffic accident, vessels can rupture and cause excessive blood loss. Many human tumors recruit local vessels to transfer nutrition for their growth. Also, shrinking or dysfunction of vessels is a common cause of body necrosis. Coronary arteries, the vessels carrying blood to the heart, can narrow or be blocked by accumulation of fat and cholesterol, commonly referred to as plaque, which can lead to the most common form of heart disease, Coronary Artery Disease (CAD). CAD is a leading cause of death among both men and women in the United States and Europe. [0003] Currently, bypass surgery is a common solution for various vessel problems. Healthy vessels removed from a human body or artificial vessels are used to act as grafts that carry blood flow around a damaged vessel. The grafts bypass the rupture or blockage and resume the normal circulation. However, drawbacks concerning this type of surgery are obvious. The graft may rupture or clog again and insufficient healing at the incision area can cause leakage and/or inflammation. Also, the removal of vessels from the human body for grafting requires significant surgery. Further, vessels from other origin, typically pig or cattle donors, is objectionable under certain religious guidelines. Current artificial vessels are chemically synthesized from two kinds of materials: polyester and expanded polytetrafluoroethylene (ePTFE). However, plastic artificial vessels made of polyester or expanded polytetrafluoroethylene (ePTFE) are often not durable and are prone to crack several years after the initial surgery. Titanium can be used as a robust supporting material for larger grafts. These artificial vessels are used mainly in bypass surgeries, but due to their disadvantages, including the possibility of cracking and/or leakage at the incision location, they are not widely used in bypass surgeries. Bypass surgeries are still dominated by natural vessel transplants which, as noted above, have certain disadvantages.

[0004] Stents are alternatives to bypass surgery for CAD patients. A stint may be in the form of either an expandable wire or a perforated tube that can be inserted in a damaged vessel to prevent or counteract a blockage. However, the long term safety of stents has not been confirmed and many physicians have proposed that approximately 20% of stent patients should receive bypass surgery rather than a stent.

[0005] Accordingly, a need exists for an improved artificial vessel.

SUMMARY OF THE INVENTION

[0006] The present invention overcomes various disadvantages of the prior art.

[0007] In one embodiment, the invention is directed to an artificial vessel comprising elastic protein and vessel growth stimulator capable of stimulating vessel endothelial cell growth. In another embodiment, the invention is directed to an artificial vessel comprising a tube formed of elastic protein and at least one element attached to the tube for physically stimulating vessel endothelial cell growth. In yet a further embodiment, the invention is directed to an implant kit which comprises an artificial vessel as described.

[0008] An additional embodiment of the invention is directed to methods of manufacturing an artificial vessel using recombinant techniques. Specifically, in one embodiment, the method of manufacturing an artificial vessel comprises inducing expression of an elastic protein-vessel growth stimulator fusion protein from a fusion gene of an elastic protein and a vessel growth stimulator.

[0009] The artificial vessels, kits and methods according to the present invention provide various advantages in production, use and/or durability over embodiments of the prior art.

Additional embodiments and advantages of the present invention will be apparent in view of the detailed description set forth herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The detailed description will be more fully understood in view of the accompanying drawings in which:

[0011] Figure 1 shows one schematic embodiment of a material for an artificial vessel according to the invention wherein resilin, an exemplary elastic protein which acts like a molecular spring, and angiogenin, an exemplary vessel growth stimulator for growth of vessel endothelial cells, are combined in a fusion protein;

[0012] Figure 2 schematically shows an embodiment of an artificial vessel implanted to bypass a blockage;

[0013] Figure 3 schematically shows various combination strategies of elastic protein and vessel growth stimulator according to the present invention; and

[0014] Figure 4 schematically shows another combination strategy of elastic protein and vessel growth stimulator according to the present invention.

DETAILED DESCRIPTION

[0015] The present invention is directed to artificial vessels comprising elastic protein which serves as a supporting material and vessel growth stimulator to induce the growth of natural vessels upon implant of the vessel within a body. The vessels are mainly, but not restricted to be, used for conveying blood flow, and may be in the form of an artery, a vein or a capillary. It is capable to encompass different shapes and, in one embodiment, the vessel is in the form of a tube. A tube is the most common choice to convey blood flow.

[0016] The artificial vessel comprises a combination of elastic protein and vessel growth stimulators, and various combination strategies may be employed. For example, in one embodiment, as shown in Figure 2, the elastic protein can be used to construct an artificial vessel tube (1) while vessel growth stimulator (2) can be linked to one or both ends of the tube. Alternatively, the vessel growth stimulator could be coated on the inner surface and/or outer surface of an elastic protein tube, either continuously or discretely (i.e., discontinuously), as shown in Figure 3. Alternatively, the vessel growth stimulator may be used to construct a tube together with elastic protein as shown in Figure 4. Different methods can be utilized to combine the elastic protein and the growth stimulator in this manner. Protein fusion is a common biological technique used to combine multiple proteins and may be used in one embodiment herein. Alternatively, various chemicals can be used to cross-link elastic protein and vessel growth stimulator. Suitable crosslinking chemicals include, but are not limited to, sulfosuccinimidyl 4-[W-maleimidomethyl] cyclohexane-1-carboxylate, l-ethyl-3-[3- dimethylaminopropyl] carbodiimide hydrochloride, and the like. Such cross-linkers may use covalent bonding, ionic bonding, hydrogen bonding, electrostatic interaction and/or surface attachment to link different components. Polymerization is another alternative for combining elastic protein and vessel growth stilumator. Depending on the material, various polymerization techniques may be used. Some elastic proteins, such as resilin, have been shown to be polymerizable by ultraviolet (UV) radiation and such techniques may be employed herein. [0017] Elastic proteins have the ability to undergo significant reversible deformation without rupture. Such materials are advantageous for artificial vessels in view of their flexibility and durability. Examples of elastic proteins used in the inventive artificial vessels include, but are not limited to, resilin, titin, elastin, fibrillin, spider silk, byssus and abductin from bivalve mollusks, glutenin, gliadin and collagen.

[0018] As shown in Figure 1, the elastic protein, for example resilin, is shown as a "spring" and is used to construct a tube-shape graft as shown in Figure 2. A unique feature of the artificial vessels of the present invention is the combination of the elastic protein with the vessel growth stimulator, thereby providing the ability to stimulate the growth of new vessels (angiogenesis). This can be used to prevent leaking at locations where a vessel graft is linked to the natural vessel. As a great benefit for patients, a major risk of vessel leakage is reduced. In order to introduce the angiogenic activity, one or more vessel growth stimulators can be fused or linked to the elastic material and function at the ends of the graft as described in Figures 1 and 2, or can be provided as a coating as shown in Figure 3. In the further embodiment of Figure 4, the elastic protein and vessel growth stimulator are combined in a single material. An example of an embodiment of the artificial vessel according to the invention as shown in Figure 4 using resilin and angiogenin is described in Examples 1 - 6.

[0019] Elastic proteins are known in the art and occur in a wide range of biological systems where they have evolved to fulfill precise biological roles. The best known elastic proteins include proteins in vertebrate muscles and connective tissues, such as titin, elastin and fibrillin, and spider silks. However, other examples include byssus and abductin from bivalve molluscs, resilin from arthropods, glutenin, gliadin, and collagen. The elastic proteins may be native or recombinant, or may comprise modifications or derivatives of the indicated proteins. Fusion proteins of the indicated proteins may also be employed in the artificial vessels; as demonstrated in the examples, an artificial vessel may be formed of a fusion protein of an elastic protein and a vessel growth stimulator.

[0020] In general, elastic proteins include proteins which exhibit elasticity and/or stretchiness. Elasticity refers to the ability of the protein to undergo significant deformation, i.e., extension, without rupture and loss of energy, before returning to its original state when the external stress causing deformation is removed. Within specific embodiments, the elastic proteins may be extended at least about 20%, preferably at least about 30%, with less than about 20%, preferably less than about 10%, permanent set. Stretchiness refers to the ability to stretch to a large degree with little force and low stiffness. Different proteins may share different characteristics regarding their elasticity and stretchiness. For example, the combination of high resilience, large strains and low stiffness is characteristic of rubber- like proteins such as resilin and elastin that function in the storage of elastic- strain energy, while collagen provides exceptional energy storage capacity but is not very stretchy. Given this range of properties and functions, elastic proteins suitable for use herein include a wealth of various chemical structures. [0021] The ability of proteins to exhibit rubber- like elasticity relates to their structure. Rubber-like materials must satisfy certain criteria: the individual components must be flexible and conformationally free, so that they can respond quickly to the applied stress, and they must be cross-linked to form a network to distribute the stress throughout the system. These crosslinks can be covalent or non-covalent. Thus, the elastic properties of proteins are influenced by the nature of the elastomeric domains, their size and the degree of cross-linking. Elastomeric proteins are widely distributed in the animal kingdom; several have been characterized in detail. Due to their chemical and physical characteristics (non-globular nature, insolubility, cross- linking etc.), detailed characterization of elastic proteins can be difficult. More recently, gene sequences have become available that have allowed sequence comparisons to be made and structure-function relationships to be studied. Most of the representative elastic proteins have distinct domain structures, with at least one domain consisting of elastomeric repeat motifs and other non-elastic domains where cross-links can be formed. Exceptions to this are resilin and abductin where cross-links occur within the elastic repeat motifs. The presence of regularly repeated sequences implies the formation of a regular structure. Although the direct determination of the structures of elastic proteins has proved problematic, the limited information that is available indicates that the repetitive sequences do form regular structures and that these may be important in the elastic mechanisms. The sequences and structures of the different elastomeric proteins would imply that there may be differences in their elastic mechanisms.

[0022] The rubber-like proteins, resilin and elastin, both exhibit reversible deformation with high resilience. Additionally, both resilin and elastin are highly stretchy, to maximal extensions in excess of 100%, with a very low modulus of elasticity. This suite of properties indicates that key functions of resilin and elastin are to provide low stiffness, high strain, and efficient elastic- energy storage components in animal devices.

[0023] Resilin was first described by Weis-Fogh, "A rubber-like protein in insect cuticle," /. Exp. Biol., 37: 887-907 (1960). It has been found in a wide array of insect specialized regions including cuticle, wings, jumping legs and so on. Resilin has been acclaimed as one of the most elastic materials known, with an elastic efficiency of 97%.

[0024] Elastin is a major extracellular matrix protein of large arteries such as the aorta, imparting characteristics of extensibility and elastic recoil. Elastin consists of approximately 36 domains with alternating hydrophobic and cross-linking characteristics. It has been suggested that these hydrophobic domains, predominantly containing glycine, proline, leucine and valine, often occurring in tandemly repeated sequences, are responsible for the ability of elastin to align monomeric chains for covalent cross-linking (See Keeley et al, "Elastin as a self-organizing biomaterial: use of recombinantly expressed human elastin polypeptides as a model for investigations of structure and self-assembly of elastin." Philos Trans R Soc Lond B Biol ScL 357(1418): 185-189 (2002)). It is found in blood, lungs, and skin, and functions in association with collagen in vertebrate connective tissue where soft, reversible elasticity is required. In addition, elastin is a major component of arteries, where its stretchiness and ability to store elastic- strain energy allow arteries to smooth the pulsatile flow of blood from the heart, lowering peak blood pressure, reducing the mechanical workload of the heart, and maintaining a relatively steady flow of blood through tissues. (See Gosline, et al "Elastic proteins: biological roles and mechanical properties," Philos Trans R Soc Lond B Biol ScL, 357(1418):121-132 (2002)). [0025] Abductin is a natural elastomer that serves as the primary building block for the abductor ligament in bivalves. It is also a protein rubber like elastin but can maintain its resilience at low temperature (See Denny et al, "Jet propulsion in the cold: Mechanics of swimming in the Antarctic scallop Adamussium colbecki " J Exp Biol, 209(Pt 22):4503-4514 (2006)). Abduction presents in the inner hinge ligament of bivalve molluscs, acting as an elastic pivot that antagonizes the action of the adductor muscle. It also acts as an energy store that opens the shell when the adductor muscle relaxes. In scallops, this action has developed into a swimming mechanism, allowing them to swim a few meters at a time by opening and closing their shells approximately four times per second (See Tatham et al, "Comparative structures and properties of elastic proteins," Philos Trans R Soc Lond B Biol ScL, 357(1418):229-34 (2002)). In general terms, the main sequence feature of abductin is the presence of many repeating sequences, all of them containing glycyl residues, in a similar manner to elastin. Amino acid analyses of abductin derived from the swimming scallop, Placopecten magellanicus, revealed the presence of three prominent amino acids: glycine, methionine, and phenylalanine. In addition, the primary sequence of Argopecten abductin shows the presence of a repeating pentapeptide sequence, FGGMG, throughout the molecule. At lower scale, tetrapeptides such as MGGG and GGMG and tripeptides such as FGG are also repeated. At higher scale, the decapeptide FGGMGGGNAG and the nonapeptide GGFGGMGGG seem to be the major repeating sequences (See Bochicchio et al, "Synthesis of and structural studies on repeating sequences of abductin," Macromol Biosci., 5(6):502-511 (2005)).

[0026] Other elastic proteins suitable for use herein show different degrees in elasticity and stretchability. Collagen is a main protein of connective tissue in animals and the most abundant protein in mammals, making up about 25% of the total protein content. The tropocollagen or "collagen molecule" subunit is a rod about 300 nm long and 1.5 nm in diameter, made up of three polypeptide strands, each of which is a left-handed helix. These three left-handed helices are twisted together into a right-handed coil, a triple helix, a cooperative quaternary structure stabilized by numerous hydrogen bonds. Tropocollagen subunits spontaneously self-assemble, with regularly staggered ends, into even larger arrays in the extracellular spaces of tissues. There is some covalent crosslinking within the triple helices, and a variable amount of covalent crosslinking between tropocollagen helices, to form the different types. A distinctive feature of collagen is the regular arrangement of amino acids in each of the three chains of these collagen subunits. The sequence often follows the pattern Gly-X-Pro or Gly-X-Hyp, where X may be any of various other amino acid residues. Gly-Pro-Hyp occurs frequently. This kind of regular repetition and high glycine content is found in only a few other fibrous proteins, such as silk fibroin. Such high glycine and regular repetitions are usually rare in globular proteins. Chemically-reactive side groups are not needed in structural proteins as they are in enzymes and transport proteins. The high content of Pro and Hyp rings, with their geometrically constrained carboxyl and (secondary) amino groups, accounts for the tendency of the individual polypeptide strands to form left-handed helices spontaneously, without any intrachain hydrogen bonding. [0027] Collagen fibrils are collagen molecules packed into an organized overlapping bundle. Collagen fibers are bundles of fibrils. Collagen fibers seen in tendons are very elastic but not very stretchy or soft. Collagen fibres are frequently arranged in parallel with elastin fibers to form connective tissues, and in these circumstances the 'stretchy' elasticity of the tissue is due primarily to the elastin (Gosline et al, supra).

[0028] In bone, entire collagen triple helices lie in a parallel, staggered array. Gaps between the ends of the tropocollagen subunits (approximately 40 nm) probably serve as nucleation sites for the deposition of long, hard, fine crystals of the mineral component, which is (approximately) hydroxyapatite, Ca₅(PO₄)₃(OH), with some phosphate. It is in this way that certain kinds of cartilage turn into bone. Collagen gives bone its elasticity and contributes to fracture resistance. [0029] Mussel byssus threads and spider dragline silks are elastic proteins of considerable strength and stiffness and are remarkably stretchy. The fibre of the mussel byssus is used to attach the animal to rocks in the wave-swept marine intertidal zone. Whole byssal fibers stretch by about 100% before breaking, but the origins of the whole-fibre behaviour are complex because the fibre is composed of two segments, a distal segment that makes up about 80% of the fibre and a short, proximal segment that makes up the remainder of the fibre (Gosline et al, supra). [0030] Fibrillin is a glycoprotein, which is essential for the formation of elastic fibers found in connective tissue. It forms the structural framework of a unique and essential class of extracellular microfibrils that endow dynamic connective tissues with long-range elasticity (Kielty et al, "Fibrillin: from microfibril assembly to biomechanical function," Philos Trans R Soc Lond B Biol ScL 357(1418):207-217 (2002)).

[0031] The giant muscle protein titin, also known as connectin, is a roughly 30,000 amino acid long filament which plays a number of important roles in muscle contraction and elasticity. The I- band region of titin, largely composed of immunoglobulin-like (Ig) domains, is believed to be responsible for the molecule's extensibility and passive elasticity.

[0032] The wheat gluten proteins correspond to the major storage proteins that are deposited in the starchy endosperm cells of the developing grain. These form a continuous proteinaceous matrix in the cells of the mature dry grain and are brought together to form a continuous viscoelastic network when flour is mixed with water to form dough. Gluten, comprising roughly 78 to 85 percent of total wheat endosperm protein, is a very large complex composed mainly of polymeric (multiple polypeptide chains linked by disulphide bonds) and monomeric (single chain polypeptides) proteins known as glutenins and gliadins, respectively. Glutenins confer elasticity, while gliadins confer mainly viscous flow and extensibility to the gluten complex. One group of gluten proteins, the HMM subunits of glutenin, is particularly important in conferring high levels of elasticity strength. These proteins are present in HMM polymers that are stabilized by disulphide bonds and are considered to form the 'elastic backbone' of gluten. However, the glutamine-rich repetitive sequences that comprise the central parts of the HMM subunits also form extensive arrays of interchain hydrogen bonds that may contribute to the elastic properties via a 'loop and train' mechanism. Genetic engineering can be used to manipulate the amount and composition of the HMM subunits, leading to either increased strength or other changes in gluten structure and properties.

[0033] Other elastic proteins are known in the art and are suitable for use in the artificial vessels of the invention.

[0034] In general, vessel growth includes two different processes. Arteriogenesis is the process of enlargement and/or elongation of existing arterial vessels. Angiogenesis is the process of the formation of new capillaries from existing capillaries. A vessel growth stimulator as included in the artificial vessels of the invention may stimulate arteriogenesis and/or angiogenesis. Examples include but are not limited to, biological growth factors, growth stimulation chemicals, and physical stimulation.

[0035] Examples of biological growth factors include, but are not limited to, angiogenin, vascular endothelial growth factor (VEGF) family, fibroblast growth factor (FGF) family, matrix metalloproteinase (MMP), monocyte chemoattractant protein, platelet-derived growth factor, fibronectin and DII4, receptors thereto, and domains, modifications and/or functional fragments thereof. These biological growth factors have different mechanisms to stimulate the growth of cells, but are effective to stimulate the growth of vessel endothelial cells responsible for forming a vessel.

[0036] Angiogenin is a small polypeptide which is implicated in the formation of new blood vessels, so called angiogenesis. It is an approximately 14,124 Da soluble protein and is a potent inducer of new blood vessel formation in vivo (See Demetres, "Refined Crystal Structures of

Native Human Angiogenin and Two Active Site Variants: Implications for the Unique

Functional Properties of an Enzyme Involved in Neovascularisation During Tumour Growth,"

MoI. Biol, 285:1209-1233 (1999) and Tello-Montoliu, "Angiogenin: a review of the pathophysiology and potential clinical applications," Journal of Thrombosis and Haemostasis, 4:1864-1874 (2006)). Physiologically, angiogenin is induced during inflammation and wound healing processes. In animal tests, angiogenin has been injected in a rabbit knee and shown to enhance healing of menisci (Tello-Montoliu, supra). Angiogenin or RNase 5 is a member of the ribonuclease (RNase) super family, a group of enzymes that catalyze the cleavage of nucleotides in RNA. It is made up of a 123 amino acid chain, its corresponding nucleotide sequence shows 33% sequence identity and 65% homology with pancreatic RNase 1 (RNase A). Angiogenin's RNase activity is directed towards 28S and 18S rRNA and the resulting product, followed by the cleavage of the RNA, is usually in the length of 100-500 nucleotides. [0037] Angiogenin's structure is known from high-resolution crystal structures. It has a kidney- shaped tertiary fold, divided into two lobes. The first lobe comprises four antiparallel β-strands in a sandwich structure. The second lobe is made up of a β-sheet formed by three antiparallel β-strands packed between helixes. There are three disulphide bonds, one in lobe one and the other two in lobe two (Demetres, supra). Angiogenin differs from all other members of the RNase superfamily in the absence of a fourth disulphide bond. While not intending to be limited by theory, it is suggested that the absence of this disulphide bond contributes to the process of angiogenesis, i.e., the stimulation of endothelial cells to form new vessels (Tello- Montoliu, supra).

[0038] Angiogenin's ribonuclease activity needs to be triggered in order for angiogenin to participate in angiogenesis. While not intending to be bound by theory, the proliferation of endothelial cells is likely to occur after angiogenin binds to an endothelial cell surface receptor and undergoes a conformational change to allow binding with RNA. A 170-kDa protein has been identified as a potential candidate as the angiogenin receptor located on the endothelial cell surface. The receptor-angiogenin complex is translocated to the nucleus and accumulated in the nucleolus, which is thought to be essential for the angiogenin's angiogenic activity. When inside the nucleolus, angiogenin may bind to DNA and thus participate in the regulation of gene expressions. While again not intending to be bound by theory, in support of vessel maturation, it is also believed that angiogenin may bind to other known specific receptor molecules such as fibroblast growth factor (FCF) (Tello-Montoliu, supra).

[0039] Members of the vascular endothelial growth factor, VEGF, family are crucial regulators of neovascularization and are characterized by a cysteine knot motif. The knot is formed of two disulfide bridges with a third disulfide bond passing through them. VEGF specifically bind cellular receptor tyrosine kinases VEGFR-I, VEGFR-2, and VEGFR-3. The VEGF family currently consists of seven members: VEGF-A, VEGF-B, placenta growth factor (PIGF), VEGF-D, virial VEGF (also known as VEGF-E) and snake venom VEGF (also known as VEGF-F). Several of the members are generated by alternative exon splicing. The splicing isoforms exhibit various properties, particular in binding to co-receptor neuropilins and heparin. [0040] The biologic function of VEGF is mediated through binding to kinase receptors. Five receptors have been identified to date. Three belong to the receptor tyrosine kinase (RTK) family and are called Fms-like tyrosine kinase- 1, kinase insert domain-containing receptor (KDR, VEGFR-2) and Flt-4. The others are non-tyrosine kinase-type receptors neuropilin-1 and neurophilin-2, which are believed to function as co-receptors for some VEGF subtypes and their isoforms. Each VEGF binds selectively to some of these receptors and together they stimulate angiogenesis (See Luca Domenico D'Andrea, "Peptide-based Molecules in Angiogenesis," Chem Biol Drug Des., 67:115-126 (2006) and Yamazaki et al "Molecular and functional diversity of vascular endothelial growth factors," Molecular Diversity, 10: 515-527 (2006)). Today, VEGF are, among other things used as a therapeutic target and diagnostic tool for cancer (See Schneider et al, "Drug insight: VEGF as a therapeutic target for breast cancer," Nat Clin Pract Oncol, 4(3):181-189 (2007)).

[0041] Fibroblast growth factor (FGF) is another family of biological growth factors. A number of FGF, including FGFl and FGF2, have been shown to exhibit angiogenic potential in vitro and in vivo. FGF exert their biological activities by binding to high affinity tyrosine kinase FGFRs on the surface of target cells. FGFs are pleiotropic factors acting on different cell types, including endothelial cells, following interaction with heparin- sulfate proteoglycans (HSPGs) and tyrosine kinase FGF receptors (FGFRs). The FGF family members form a complex in endothelial cells that recapitulates several aspects of the in vivo angiogenesis process, including the modulation of endothelial cell proliferation, migration, protease production, integrin and cadherin receptor expression, and intercellular gap-junction communication (Presta et al, "Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis," Cytokine & Growth Factor Reviews, 16:159-178 (2005)).

[0042] FGF has been tested as a potential substance for therapeutic angiogenesis as a potential treatment for patients with coronary (CAD) or peripheral (PAD) artery injury. The aim of such therapy is to restore and maintain tissue perfusion by increasing the number of collateral blood vessels within ischemic territories following delivery via methods including intravenous, intracoronary, intramyocardinal and intrapericardial routes to administer angiogenic factors (Presta et al, supra).

[0043] Another biological factor for angiogenesis is matrix metalloproteinase (MMP). MMP helps degrade the proteins that keep the vessel walls solid. This proteolysis allows the endothelial cells to escape into the interstitial matrix as seen in sprouting angiogenesis. Inhibition of MMPs prevents the formation of new capillaries. These enzymes are highly regulated during the vessel formation process because wanton destruction of the extracellular matrix would destroy the integrity of the microvasculature.

[0044] Monocyte chemoattractant protein- 1 (MCP-I) is a stimulator of collateral artery growth and has been shown to increase collateral artery conductance in rabbits and pigs (see Seidler, "Short-term intra- arterial infusion of monocyte chemoattractant protein- 1 results in sustained collateral artery growth," Journal of Cardiovascular Pharmacology and Therapeutics, 12(l):61-68 (2007)).

[0045] Platelet-derived growth factor (PDGF) is known to regulate cell growth and division. In particular, it plays a significant role in blood vessel formation (angiogenesis). Chemically, platelet-derived growth factor is dimeric glycoprotein composed of two A or two B chains. There are five different isoforms of PDGF that activate cellular response through two different receptors. Known ligands include A (PDGFA), B (PDGFB), C (PDGFC) and D (PDGFD) and an AB heterodimer and receptors alpha (PDGFRA) and beta (PDGFRB).

[0046] Fibronectin regulates capillary endothelial cell growth in the presence of angiogenic mitogens while DII4 is a recently discovered material with important angiogenic properties similar to VEGF. Both of these materials may be used in the inventive artificial vessels. [0047] Chemical stimulators for the growth (including migration) of endothelial cells may also be employed. Examples, include, but are not limited to, arachidonic acid epoxygenase metabolites (See Wang, et al, "Arachidonic acid epoxygenase metabolites stimulate endothelial cell growth and angiogenesis via mitogen-activated protein kinase and phosphatidylinositol 3- kinase/akt signaling pathways," Journal of Pharmacology and Experimental Therapeutics, 314: 522-532 (2005)), alginate oligosaccharides (See Kawada et al, "Alginate oligosaccharides stimulate VEGF-mediated growth and migration of human endothelial cells," Arch Dermatol Res., 291: 542-547 (1999)), uridine, thymidine, amyloid peptides (See Cantara et al, "Physiological levels of amyloid peptides stimulate the angiogenic response through FGF-2," FASEB Journal, 18: 1943-1945 (2004)), sphingosine-1 -phosphate, lysophosphatidic acid (See Panetti et al, "Sphingosine-1 -phosphate and lysophosphatidic acid stimulate endothelial cell migration," Arterioscler Thromb Vase Biol., 20: 1013-1019 (2000)), and oxidized phospholipids (See Bochkov et al, "Oxidized phospholipids stimulate tissue factor expression in human endothelial cells via activation of ERK/EGR-1 and Ca⁺⁺/NFAT," Blood, 99(1): 199-206 (2002)). The mechanism of these chemicals are different. They may, for example, act as the metabolites of cell growth or stimulate the production of biological growth factors in endothelial cells or surrounding cells. As compared with the biological growth factors, these chemicals are normally smaller in size and can be cross-linked with elastic protein using different chemical cross-linkers. [0048] In addition to biological and chemical growth stimulators, physical stimulation can also be employed as a vessel growth stimulator. Vibration at a specific frequency may stimulate the growth of endothelial cells. Other physical parameters such as temperature, humidity and the like may also be employed as vessel growth stimulators. Devices producing such physical stimulation can then be attached to an artificial vessel portion formed of elastic protein. [0049] The present invention is also directed to kits for use, for example, in artificial vessel implant procedures. The kits may contain one or more artificial vessels, for example of different diameters, lengths and the like. Optionally, the kits may include instructions for implanting an artificial vessel in vivo in a human or animal.

[0050] The following examples demonstrate specific embodiments of the invention but should not be construed as limiting the scope of the invention defined by the claims. EXAMPLES

Example 1: Cloning of the resilin gene from Drosophila melanogaster [0051] According to WO 2004/104042 Al, the first exon of the resilin gene can be amplified from the genomic DNA of Drosophila melanogaster to produce recombinant resilin via PCR. A pair of PCR primers is designed from the known DNA sequence of Drosophila resilin gene (CG15920). The forward primer contains a Sful restriction site and 6x His tag. The reverse primer contains an EcoRI restriction site. The PCR product is confirmed by DNA sequencing and cloned into the SfuI/EcoRI site of a Pichia pastoris expression vector pPICZ A (Invitrogen). [0052] The PCR primers are Res-forw (forward) and Res-rev (reverse) and their sequences are:

Res-forw:

CCTTCGAACACCATCACCATCACCATCCGGAGCCACCAGTTAACTCGTATC TACC (SEQ ID NO: 1) Res-rev:

CCGAATTCCTATCCAGAAGCTGGGGGTCCGTAGGAGTCGGAGGG (SEQ ID NO: 2)

The respective restriction sites are underlined.

Example 2: Cloning of the angiogenin gene from human lung cancer cell line A549 [0053] Human angiogenin gene is amplified by reverse transcription PCR from the human lung cancer cell line A549 (Lu et α/.2000). Total RNA is isolated using GenElute Mammalian

Total RNA kit (Sigma- Aldrich) and then is used to synthesize the first-strand cDNA. For the reverse transcription PCR, the synthesized cDNA is used as the template. The forward PCR primer contains an EcoRI restriction site while the reverse primer contains a PmII site. The PCR product is confirmed by DNA sequencing and cloned into the EcoRI/Pmll site of pPICZ A plasmid.

[0054] PCR primers are Ang-forw (forward) and Ang-rev (reverse) and their sequences are: Ang-forw: GAATTCCGGACGACGGAAAATTGACTGATCCAAGTG (SEQ ID NO: 3)

Ang-rev: CACGTGCAGGATAACTCCAGGTACACACACTTCCTG (SEQ ID NO:

4)

The respective restriction site is underlined.

Example 3: Construction of resilin-angiogenin fusion gene [0055] The resilin and angiogenin cDNAs from the respective pPICZ A plasmids are fused together by splicing by overlap extension (SOE) PCR. Hybrid primers are used for this purpose. The name and sequence of these primers are: [0056] h-Res-forw:

CCTTCGAACACCATCACCATCACCATCCGGAGCCACCAGTTAACTCGTATC

TACC (SEQ ID NO: 1)

Underlined: Sful site h-Res-rev:

CAGGAAGTGTGTGTACCTGGAGTTATCCTGGAATTCCTATCCAGAAGCTGG

GGGTCCGTAGGAGTCGGAGGG (SEQ ID NO: 5)

Italic: 3'-Angiogenin region; underlined: EcoRI site [0057] h-Ang-forw:

CCCTCCGACTCCTACGGACCCCCAGCTTCTGGATAGGAATTCCGGACGACG GAAAATTGACTGATCCAAGTG (SEQ ID NO: 6) Italic: 5'-Resilin region; underlined: EcoRI site h-Ang-rev:

CACGTGCAGGATAACTCCAGGTACACACACTTCCTG (SEQ ID NO: 4) Underlined: PmII site [0058] h-Res-forw and h-Res-rev are used in the 1^st PCR to produce a resilin sequence with a

3' angiogenin partial region overhang connected by an EcoRI restriction site. In the 2^nd PCR, h- Ang-forw and h-Ang-rev are used to amplify an angiogenin sequence with a 5' resilin partial region overhang connected by an EcoRI restriction site. The products from the first two PCR are digested by EcoRI enzyme and linked together by DNA ligase. This serves as the template in the 3^rd PCR using primer h-Res-forw and h-Ang-rev. The final hybrid resilin-angiogenin fusion gene is cloned into the Sful/Pmll site of pPICZ A vector.

Example 4: Test expression and purification of the first exon of the resilin gene and resilin- angiogenin fusion gene [0059] The pPICZ A plasmid containing the first exon of the Drosophila resilin gene is linearized by Sad digestion overnight. 10 μg of linearized plasmid is then used to transform the competent Pichia pastoris cells. Two different Pichia strains, XL33 and KM71H, are used to compare the efficiency of transformation and resilin expression. EasyCom™ Pichia transformation kit (Invitrogen) is used for transformation and the transformants are spread on YPD plates containing 100 μg/ μl Zeocin. The cells that have incorporated the plasmid can survive in the existence of Zeocin and are picked up and grown in 100 ml of MGY media until reaching the log phase. The cells are then transferred to 20 ml of MM media and supplemented with 0.5% methanol every 24 hours to induce the expression of recombinant resilin gene. Cell samples from different time points of induction, such as 0 hr, 6 hr, 24 hr, 48 hr, 72 hr and 96 hr are collected. Proteins are extracted and the time-course expression of resilin is detected by SDS- PAGE and western blot using antibody against resilin or the 6x His tag. The efficiency of resilin expression from different colonies is compared and the most efficient one is used to express resilin on a large scale. The test expression of resilin-angiogenin fusion gene is carried out in the same way as described for resilin.

[0060] Both the recombinant resilin and resilin-angiogenin gene have a 6x His tag, therefore, the expressed proteins can be purified by Immobilized Metal Affinity Chromatography (IMAC). Sepharose™ fast flow medium (GE Healthcare) is first charged with 0.2 M NiCl₂ to form a chelate and then packed into a column. Protein samples are applied to the column. Proteins having the 6x His tag will bind to the column and the unbound proteins are washed away by low strength ionic buffer such as 40 mM imidazole. The bound protein is then eluted from the column by high strength ionic buffer such as 300 mM imidazole. The eluted proteins are concentrated and analyzed by SDS-PAGE and western blot to assess purity.

Example 5: Large scale expression and purification of resilin and resilin-angiogenin fusion proteins [0061] Two liters of MGY media is inoculated with 1 ml of an overnight YPD culture of selected colonies. The cells are grown with vigorous shaking and aeration on a rotary shaker (250 rpm) at room temperature until the ODβoo reaches 8 - 11. The cells are then harvested by centrifugation (2,000 rcf 5 min at room temperature). The cell pellets are resuspended in 400 ml of MM media and grown with vigorous shaking and aeration on a rotary shaker (250 rpm) at room temperature for 3-5 days. Fresh methanol is added to the culture every 24 hours to a final concentration of 0.5%.

[0062] The cells are pelleted down by centrifugation (2,500 rcf 5 min at room temperature) and resuspended to final volume of 45 ml using cell breaking buffer (50 mM Tris, 100 mM NaCl, 10% glycerol, pH 7.8). The cell suspension is mixed with an equal volume of glass beads (ø = 0.5 mm) and disrupted in a mechanical breaker. The glass beads are filtered by vacuum and the cell lysate is centrifuged at 2,500 rcf for 15 min at 4⁰C. The supernatant is collected and supplemented with 1% Triton X-100, 0.5% deoxycholate and 5 mM β-mercaptoethanol. The supernatant is incubated at 4⁰C with gentle shaking for 1 hr to solubilize proteins. After that, the soluble fraction is separated by centrifugation at 15,000 rcf for 15 min at 4⁰C. [0063] The soluble fraction is incubated with 2 ml of Ni ⁺-charged sepharose medium for 1 hr at 4⁰C. The resin is pelleted down by centrifugation at 1,000 rcf for 1 min at 4⁰C and packed into a column which is washed with 4 ml of washing buffer (50 mM Tris, 0.5 M NaCl, 10% glycerol with 40 mM imidazole) three times. The bounded proteins are eluted by 4 ml of elution buffer (50 mM Tris, 0.5 M NaCl, 10% glycerol with 300 mM imidazole) three times. Wash and elution fractions are collected and 10 μl aliquots of each fraction are analyzed by SDS-PAGE and western blot using antibody against resilin or 6x His tag.

[0064] The elution fractions are combined and concentrated using a Millipore/Amicon ultrafiltration tube (cut off 10 kDa). The proteins are dialysed using a 10 kDa cut off membrane, overnight against 5 liters of 50 mM Tris and 50 mM NaCl (pH 7.5). At this point, the proteins are ready for further experiments.

Example 6: UVB radiation crosslinking of resilin and resilin-angiogenin fusion proteins [0065] According to WO 2004/104042 Al, resilin can be crosslinked by UVB radiation to form a polymer. The mechanism of this crosslinking has been proposed to be related with dityrosine formation (Elvin et al, "Synthesis and properties of crosslinked recombinant pro- resilin," Nature, 437 (7061):999-1002 (2005)). The resilin-angiogenin fusion protein has the same potential as resilin to form dityrosine and may be crosslinked in the same manner. The methods are described in WO 2004/104042 Al. Briefly, the concentrated proteins are diluted in PBS to give a final concentration of 23 mg/ml. The samples are then exposed to UVB radiation using UVB tubes at room temperature for 1, 2, 4, 8, 16, 32 and 64 hours. [0066] The artificial vessels of the present invention are distinguished from current artificial vessels in, inter alia, the use of a combination of elastic protein and vessel growth stimulator to provide life-long endurance and stimulation of vessel growth processes. The elastic protein provides a supporting material with extremely strong tolerance to external and internal forces and the ability to deform reversibly, thereby reducing or even eliminating the cracking problems often encountered with current artificial vessels. Meanwhile, the excellent physical features of elastic proteins make it possible for the present invention to replace expansive titanium racks often required in larger grafts. The incorporated vessel growth stimulator contributes to avoiding the fatal leakage problems often encountered with current artificial vessels. [0067] Comparing current artificial vessel implants, the present invention has substantial advantages, including:

1) Facilitating growth of new vessels at an incision location during surgery to form a niche-less joint which avoids potential leakage;

2) Durability, functioning as long as patients live due to the extreme durability and flexibility of natural elastic protein;

3) Utilization of natural protein material reduces the risk of developing rejection or inflammation;

4) Withstanding large external forces without breaking, which is ideal for the removal of recurrent blockage by sonication.

The unique characteristics of the artificial vessels of the present invention provide patients with a safe and effective therapy. Advantageously, patients will typically require only one operation, without additional surgeries to eliminate leakage or blockage, which is extremely both treatment- effective and cost-effective.

[0068] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments described in the specification and/or the examples. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, additional embodiments, presently existing or later to be developed, that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention. Accordingly, the appended claims are intended to include within their scope such additional embodiments.

SEQUENCE LISTING

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<120> Artificial Vessels, Kits and Methods

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Claims

WHAT IS CLAIMED IS:

1. An artificial vessel, comprising elastic protein and vessel growth stimulator capable of stimulating vessel endothelial cell growth.

2. The artificial vessel of claim 1, wherein the elastic protein is in the form of a tube.

3. The artificial vessel of claim 2, wherein the vessel growth stimulator is provided in a coating on at least one of an inner surface and an outer surface of the tube.

4. The artificial vessel of claim 2, wherein the vessel growth stimulator is provided at one or both ends of the tube.

5. The artificial vessel of claim 1, wherein the vessel is in the form of a tube and wherein a wall of the tube comprises both the elastic protein and the vessel growth stimulator.

6. The artificial vessel of claim 1, wherein the elastic protein comprises one or more of resilin, elastin, titin, fibrillin, spider silk, byssus, abductin, glutenin, gliadin, and collagen.

7. The artificial vessel of claim 1, wherein the elastic protein comprises resilin.

8. The artificial vessel of claim 1, wherein the vessel growth stimulator comprises a biological vessel growth stimulator, one or more of angiogenin, vascular endothelial growth factor, fibroblast growth factor, matrix metalloproteinase, monocyte chemoattractant protein, platelet-derived growth factor, fibronectin, and DII4.

9. The artificial vessel of claim 8, wherein the biological vessel growth stimulator comprises a chemical vessel growth stimulator.

10. The artificial vessel of claim 1, wherein vessel growth stimulator comprises a chemical vessel growth stimulator.

11. The artificial vessel of claim 10, wherein the chemical vessel growth stimulator comprises one or more of arachidonic acid epoxygenase metabolites, alginate oligosaccharides, uridine, thymidine, amyloid peptides, sphingosine-1 -phosphate, lysophosphatidic acid, and oxidized phospholipids.

12. The artificial vessel of claim 1, wherein the elastic protein comprises resilin and the vessel growth stimulator comprises angiogenin.

13. The artificial vessel of claim 1, comprising recombinant elastic protein-vessel growth stimulator fusion protein.

14. The artificial vessel of claim 11, comprising recombinant resilin-angiogenin fusion protein.

15. The artificial vessel of claim 12, wherein the protein is crosslinked.

16. An implant kit, comprising the artificial vessel of claim 1.

17. An artificial vessel, comprising a tube formed of elastic protein and at least one element attached to the tube for physically stimulating vessel endothelial cell growth.

18. The artificial vessel of claim 15, wherein the element is operable to vibrate at least one end of the tube sufficiently to stimulate vessel endothelial cell growth.

19. A method of manufacturing an artificial vessel, comprising inducing expression of an elastic protein- vessel growth stimulator fusion protein from a fusion gene of an elastic protein and a vessel growth stimulator.