Rational Engineering and Use of Angiogenic Polymers

From our discussion of the basic biology of ECM-induced angiogenesis, and of the tissue engineering of the microvasculature, it is obvious that a crossroads of both bodies of knowledge is the matter of how one may define and rationally create an angiogenic polymer. The simplest approach to this end may be to design a polymer that has all of the appropriate, proangiogenic characteristics of type I collagen or fibrin, but that lacks any of their undesirable characteristics (Table I). For example, to replicate some of the common features of type I collagen and fibrin, the polymer should assume a rigid, linear shape approximately

Table I Angiogenic Super-Polymers.

Physical characteristics

• Form a rigid, cable-like polymer

• Assume appropriate secondary and tertiary conformations

• Assume dimensions of @ 100 nm wide and > 100 mm long

• Gel at physiological temperature and pH

• Carry a net positive charge

Biologically Active Determinants

Include sites:

• For binding of appropriate *integrins, distributed 10-50nm apart, or for binding of other cell surface receptors.

• For binding of angiogenic growth factors (e.g., bFGF/VEGF)

• Necessary for polymer fibrillogenesis/assembly

Exclude binding sites for:

• Ligands that sterically disrupt EC-polymer interactions

• Proteinases, pathogens, or toxins Exclude epitopes that are:

• Immunogenic

• Pro- or anticoagulant

• Antiangiogenic

Modes of presentation to EC

• As apical or pericellular gel

• Affixed to EC substrata in stripes/islands < 10 mm wide

• Injected into tissues with vascular insufficiency/ischemia

• Incorporated into tissue equivalent scaffolds for implantation in vivo

• Genetically engineered to be expressed by perivascular cells or vascular stem cells

*Integrin-binding sites in type I collagen or collagen mimetic polymers must be triple helical.

Features and uses of angiogenic super-polymers are proposed based on common characteristics of the proangiogenic type I collagen and fibrin polymers, and on various aspects of EC biology outlined in this review. Super-polymers may include genetically engineered forms of type I collagen or fibrin, or various synthetic polymers.

100nm or greater in diameter and sufficiently long (i.e., > 100 mm) to simultaneously interact with or bridge two or more EC. The ideal polymer might carry a basic charge, because this will confer affinity for the strongly anionic character of the EC surface. Moreover, the polymer should contain ligand-binding sites for an appropriate EC surface receptor, such as the a2b1 integrin (i.e., GFPGER sequences), which should be distributed appropriately to promote cell-polymer interactions. From this discussion, such integrin-binding sites might be spaced no closer than 10 nm and no farther apart than 50 nm on the polymer. If the polymer is to be presented apically to cells, it should be capable of forming a classic gel or, if it must be affixed as stripes on a substrata on which EC will be seeded, one would limit their width to 10 mm or less to promote proper lumen formation. To create such a polymer, either recombinant type I collagen or fibrin could be used, in which unsuitable protein epitopes are deleted (e.g., Table I). For example, in the case of type I collagen, one might remove binding sites for ligands that have the potential to interfere directly, or through steric hindrance, with integrin receptor binding, such as fibronectin and the various collagen-binding proteoglycans such as decorin. Another approach would be to design polymers having no significant homologies to collagen or fibrin, but in which multiple integrin-binding sites are included at appropriate spacing intervals. Such super polymers may someday provide an ideal angiogenic template for a variety of tissue engineering and human therapeutic applications.

Glossary

Angiogenesis: Establishment of new blood vessels by branching, budding, or remodeling of the existing vasculature. Occurs during embryogenesis and in the adult (e.g., during wound healing).

Basement membrane: Extracellular matrix scaffold secreted by EC and other cell types, comprised of laminin, type IV collagen, perlecan, and nidogen, as well as other minor components that vary depending on the tissue source. The basement membrane lies in close contact with the basal side of the EC and provides structural support and functions as a barrier separating them from the underlying perivascular cells and tissue stroma.

EC: Mesenchymal cells that comprise the lining of the cavities of the heart, blood, and lymphatic vessels.

Fibrin: A polymer that forms the fibrous stroma of blood clots. It is generated during the clotting cascade by thrombin cleavage of fibrinogen, which liberates fibrin monomers that assemble in an overlapping fashion into the cable-like fibrin polymer.

Type I collagen: A ubiquitous ECM component comprised of triple-helical monomers that assemble in a staggered fashion into cable-like collagen fibrils in the extracellular space, which confer tensile strength on many tissues of the body.

References

1. Gerritson, M. E. et al. (2003). Microcirculation, 10, 63-81.

2. Weisel, J. W. (1986). Biophys. J, 50, 1079-1093.

3. Vernon, R. B. et al. (1995). In Vitro Cell Dev. Biol. 31, 120-131.

Further Reading

D'amore, P. A., and Ng, Y. S. (2002). Tales of the cryptic: Unveiling more angiogenesis inhibitors. Trends in Molec. Med. 8, 313-315.

DiLullo, G., Sweeney, S. M., Korrko, J., Ala-Kokko, L., and San Antonio, J. D. (2002). Mapping the ligand-binding sites and disease associated mutations on the most abundant protein in the human- type I collagen. J. Biol. Chem. 277, 4223-4231.

Iozzo, R., and San Antonio, J. D. (2001). Heparan sulfate proteoglycans: Heavy hitters in the angiogenesis arena. J. Clin. Invest. 108, 349-355.

Moldovan, N., and Ferrari, M. (2002). Prospects for microtechnology and nanotechnology in bioengineering of replacement microvessels, Arch. Pathol. Lab. Med. 126, 320-324. This review outlines many of the aspects of microvascular tissue engineering discussed here and provides their primary literature citations.

Mongiat, M., Sweeney, S. M., San Antonio, J. D., Fu, J., and Iozzo, R. (2003). Endorepellin, a novel inhibitor of angiogenesis derived from the c terminus of perlecan. J. Biol. Chem. 276, 4238-4249.

Somerville, R. P. T., Oblander, S. A., and Apte, S. S. (2003). Matrix metal-loproteinases: Old dogs with new tricks. Genome Biol. Accessed at: http://genomebiology.com/2003/476/216.

Sudhakar, A., Sugimoto, H., Yang, C., Lively, J., Zeisberg, M., and Kalluri, R. (2003). Human tumstatin and human endostatin exhibit distinct antiangiogenic activities mediated by av|33 and a5|31 integrins. Proc. Natl. Acad. Sci. USA 100, 4766-4771.

Sweeney, S. M., DiLullo, G. D., Slayter, S., Martinez, J., Iozzo, R. V., Lauer-Fields, C., Fields, G. B., and San Antonio, J. D. (2003). Type I collagen-induced angiogenesis requires a2|31 ligation of a single GFP*GER integrin-binding collagen sequence, possibly in concert with p38 MAPK activation and focal adhesion disassembly. J. Biol. Chem. 278, 30516-30524.

Weideman, M. P., Tuma, R. F., and Mayrovitz, H. N. (1981). An Introduction to Microcirculation, Vol. 2. New York: Academic Press. This book describes the anatomy and function of the microvasculature and provided facts cited here regarding the structure of capillary beds, average dimensions of capillaries, and the anatomy of the bat wing vasculature.

Xu, J., and Brooks, P. C. (2002). Extracellular Matrix in the Regulation of Angiogenesis in Assembly of the Vasculature and Its Regulation (R. J. Tomanek, ed.), pp. 67-95. Boston: Birkhauser Press. This article presents a comprehensive review of ECM structure and its many roles in angiogenesis, and provides primary citations for many of the facts discussed here. Although the authors categorize the ECM into the basement membrane and interstitial compartments, they do not present them as having two distinct functions in angiogenesis regulation as we have done here.

Capsule Biographies

Dr. James D. San Antonio is an Associate Professor of Medicine at Thomas Jefferson University in Philadelphia. His research focuses on elucidating mechanisms of type I collagen-induced angiogenesis; defining the role of proteoglycan-collagen interactions in connective tissue disorders; and invention of heparin and proteoglycan-binding peptides to use as pro-tamine substitutes for the neutralization of anticoagulant heparin in humans, or as carriers to target drugs to proteoglycan-rich tissues such as the vasculature. His work is supported by grants from the National Institutes of Health, the Pennsylvania American Heart Association, and the Department of Defense.

Dr. Renato V. Iozzo is a Professor of Pathology and Cell Biology, and Director of the Extracellular Matrix Program at the Kimmel Cancer Center, at Thomas Jefferson University, in Philadelphia, Pennsylvania. He is the winner of several awards and was the past Chair of the Gordon Research Conference on Proteoglycans. He is the editor of two books and has published more than 200 research articles focused on extracellular matrix, tumor growth, and angiogenesis. His current work is supported by funds from the National Institutes of Health and the Department of Defense.

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