Targeting Inflammation

target-figure1-1024x755A variety of cardiovascular and metabolic disorders, as well as responses among the critically ill surgical patient to injury and repair are all modulated by local inflammatory responses. For example, the recruitment of neutrophils, monocytes, and platelets to the arterial wall is considered a critical step in the earliest stages of atherosclerosis, as well as restenosis following angioplasty and vascular bypass. A significant body of evidence also suggests that an inflammatory cascade, which leads to cytokine-mediated stimulation of metalloproteinase expression, is an important contributing factor in aneurysm expansion. Control of inflammation and innate immune responses expand the prospects for improving clinical outcomes in a broad based group of diseases.

Selectin Antagonists

target-figure2-300x203Selectins (L, E- and P) are class of vascular endothelial molecules that play a critical role in the recruitment of leukocytes to inflamed tissue. In this regard, P-Selectin Glycoprotein-1 (PSGL-1) has been identified as the best characterized ligand for P-selectin. PSGL-1 binds to P-selectin through the interaction of core-2 O-glycan expressing Sialyl Lewis x oligosaccharide and the three tyrosine sulfate residues. Some of the challenges associated with the synthesis of PSGL-1 include obtaining the hexasaccharide component with Sialyl Lewis x portion and the synthesis of tyrosine sulfates in the peptide backbone. The presence of sulfotyrosines is critical to the binding of PSGL-1 as it has been demonstrated previously that without sulfotyrosine residues the glycopeptide will lose substantial activity. We are currently investigating chemical ligation approaches to synthesize glycosulfo peptides and respective mimics as anti-inflammatory agents.

Rele SM, Cui W, Hou S, Barr-Zarse G, Tatton D, Gnanou Y, Esko J, Chaikof EL. Dendrimer-like PEO glycopolymers exhibit anti-inflammatory properties. J Am Chem Soc 2005; 127:10132-10133.

Feng XS, Taton D, Chaikof EL, Gnanou Y. Fast access to dendrimer-like poly(ethylene oxide)s through anionic ring-opening polymerization of ethylene oxide and use of nonprotected glycidol as branching agent. Macromolecules 2009; 42: 7292-7298.

Feng X, Chaikof EL, Absalon C, Drummond C, Taton D, Gnanou Y. Dendritic carrier based on PEG: Design and degradation of acid-sensitive dendrimer-like poly(ethylene oxide)s. Macromol Rapid Comm 2011; 32:1722-1728.

Feng X, Pinaud J, Chaikof EL, Taton Y, Gnanou Y. Sequential functionalization of Janus-type dendrimer-like poly(ethylene oxide)s with camptothecin and folic acid. J Polymer Sci Part A-Polym Chem 2011; 49: 2839-2849.

Krishnamurthy VR, Dougherty A, Kamat M, Song X, Cummings RD, Chaikof EL. Synthesis of a Fmoc-threonine bearing core-2 glycan: A building block for PSGL-1 via Fmoc assisted solid phase peptide synthesis. Carbohydrate Research 2010; 345:1541-1547

Biologically Inspired Materials for Reparative Medicine and Organ Fabrication

bioinspiredThe development of bio-inspired materials will provide component building blocks for enabling advances in cell-based therapies, artificial organs, and engineered living tissues, all of which will define the evolving field of Reparative Medicine.

 

Synthetic Elastin Analogues

Elastin_1jpg-300x208Recombinant protein engineering can significantly increase protein yield over that which can be achieved by extraction of a native protein from animal tissues and offers the ability to use human amino acid sequences, so as to avoid adverse immunological responses. However, the most important impact of this technology lies in the potential to introduce precise changes in the amino acid sequence and/or to construct new proteins based upon the assembly of de novo peptide sequences or through the use of non-natural amino acids. One example is the generation of structural proteins, referred to as protein polymers that consist of sequentially repeated amino acid blocks. Typically, the incorporation of repetitive oligopeptide sequences, derived from a consideration of the primary amino acid structure of a native protein, imparts critical structural properties from the parent protein to the recombinant polypeptide. Moreover, opportunities to improve the biological, thermodynamic, and mechanical properties of the protein polymer exist through alteration of the peptide chain length, consensus repeat sequence, and the introduction of additional functional groups or oligopeptide units. For example, we have demonstrated that substituting different amino acids for those ordinarily occurring in the sequence can affect the susceptibility of the protein to proteolytic degradation or facilitate the placement of crosslinks at well-defined intervals along the polypeptide chain. It is also significant that the uniformity of macromolecular structure achieved by recombinant strategies provides exquisite control over macroscopic polymer properties, including material processability. In summary, the possibility now exists to generate synthetic polypeptides that mimic structural matrix proteins.

Elastin_2Recombinant elastin-like protein polymers (ELP) represent a promising new class of biomaterials with potential applications in drug delivery, tissue engineering, or as components of implanted medical devices. Through a multidisciplinary effort involving a diverse group of chemists and engineers, our group is designing and investigating a number of novel photochemically and virtually crosslinked elastomeric recombinant protein polymers. In particular, our studies have lead to the generation of ELP triblock copolymers, consisting of hydrophilic, elastomeric midblock sequences flanked by self-associating, hydrophobic endblocks in arranged in an ABA block format. Sequences with individual block sizes in excess of 35 kDa have resulted in protein-based biomaterials demonstrating structural polymorphism, allowing us to broadly tune material properties. By adjusting polymer sequence and processing conditions, resilient ELPs with a broad range of stiffness and strength have been formulated as films, gels, micelles, or nanofibers. Subcutaneous implants in mice have revealed that some ELPs largely resist biodegradation for at least a year following implant. Ex vivo baboon experiments have indicated that ELP coatings on the inner surface of small diameter vascular grafts dramatically reduce clotting (platelet and fibrin deposition) in the first hour of blood flow. Overall, our research has produced a series of ELPs in high yield, with molecular weights ranging from 100 to 250 kD, and with low endotoxin levels for a variety of applications in tissue engineering, drug delivery, and device design.

Nagapudi K, Huang L, McMillan RA, Brinkman W, Conticello VP, Chaikof EL. Photomediated solid-state crosslinking of an elastin-mimetic recombinant protein polymer. Macromolecules 2002; 35:1730-1737

Nagapudi K, Brinkman WT, Thomas BS, Wright ER, Conticello VP, Chaikof EL. Protein-based thermoplastic elastomers. Macromolecules 2005; 38:345-354.

Nagapudi K, Brinkman WT, Thomas BS, Ok-Park J, Sreenivasarao M, Wright E, Conticello VP, Chaikof EL. Viscoelastic and mechanical behavior of recombinant protein elastomers. Biomaterials 2005; 26:4695-4706.

Wu X, Sallach R, Haller CA, Caves JA, Nagapudi K, Conticello VP, Levenston ME, Chaikof EL. Alterations in physical crosslinking modulate mechanical properties of two-phase protein polymer networks. Biomacromolecules 2005; 6:3037-304

Sallach RE, Cui W, Wen J, Martinez A, Conticello VP, Chaikof EL. Elastin-mimetic protein polymers capable of physical and chemical crosslinking. Biomaterials 2009; 30:409-422.

Sallach RE, Cui W, Balderrama F, Martinez AW, Wen J, Haller CA, Taylor JV, Wright ER, Long RC, Chaikof EL. Long-term biostability of self-assembling protein polymers in the absence of covalent crosslinking. Biomaterials 2010; 31:779-91

Ravi S, Krishnamurthy VR, Caves JM, Haller CA, Chaikof EL. Maleimide-thiol coupling of a bioactive peptide to an elastin-like protein polymer. Acta Biomaterialia 2012; 8:627-635.

Ravi S, Haller CA, Sallach RE, Chaikof EL. Cell behavior on a CCN1 functionalized elastin-mimetic protein polymer. Biomaterials 2012; 33:2431-8

Synthetic Collagen Fiber Analogues

collagen_1Collagen comprises the major structural protein component of the extracellular matrix of higher organisms and collagen-based materials are prime candidates for tissue repair and replacement technologies. However, it remains a major challenge to emulate the unique structural and biological properties of native collagenous biomaterials in synthetic analogues. Collagens derived from animal sources are widely employed as biomedical materials although their practical utility is limited by the possible contamination of the material with pathogenic substances, such as viruses and prions, lack of sequence control, and potential immunogenicity in humans. Consequently, numerous opportunities exist for synthetic collagens in biomedical applications as extracellular matrix analogues, if the appropriate materials could be constructed that retain and expand upon the desirable properties of native collagen fibrils. The exploration of chemical and molecular genetic techniques to design and synthesize collagen-mimetic polypeptides and fibers that are competent for self-assembly into structurally defined protein fibrils is an intriguing avenue for exploration.

collagen_2We are not only involved in the synthesis of collagen biopolymer analogues but have active programs in the application of novel fabrication strategies for the design of a variety of engineered tissues, including heart valves and blood vessel substitutes. We have developed a wet spinning method for the scalable production of collagen microfiber, used to reinforce protein-based composite materials for small diameter vascular grafts and soft-tissue repair patches. In addition, we are applying variety of microfabrication techniques to generate collagen crimped-fiber composites that recapitulate native tissue architecture and biomechanics.

Huang L, Nagapudi K, Brinkman W, Apkarian R, Chaikof EL. Engineered collagen-PEO nanofibers and fabrics. J Biomaterials Sci – Polymer Ed. 2001; 12:979-994.

Brinkman WT, Nagapudi K, Thomas BS, Chaikof EL. Photocrosslinking of type I collagen gels in the presence of smooth muscle cells: Mechanical properties, cell viability and function. Biomacromolecules 2003; 4:890-895

Rele S, Song Y, Apkarian RP, Qu Z, Conticello VP, Chaikof EL. D-Periodic collagen-mimetic microfibers. J Am Chem Soc 2007; 129:14780-14787.

Caves JM, Kumar VA, Wen J, Cui W, Martinez A, Apkarian R, Coats JE, Berland K, Chaikof EL. Fibrillogenesis in continuously spun synthetic collagen fiber. J Biomed Mater Res. Part B. Appl Biomat 2010; 93:24-38.

Caves JM, Kumar VA, Xu W, Naik N, Allen MG, Chaikof EL. Microcrimped collagen fiber-elastin composites. Advanced Materials 2010; 22:2041-2044.

Membrane-Mimetic Systems

MembraneLipid-based membranes have attracted considerable attention due to their potential application as tools to probe cellular and molecular interactions and as bioactive coatings for biosensor or medical implant applications. In particular, phospholipids differing in chemical composition, degree of saturation and size have provided primary building blocks for membrane-based structures of varying geometry because of their intrinsic biocompatibility, high packing density, and propensity to form lamellar systems. Nonetheless, inherently limited stability continues to restrict the use non-covalently associated lipid membrane systems to transient or short-term applications. Prior studies by our group have led to the fabrication of supported membrane-like films by photocrosslinking of polymerizable lipids. Significantly, the ability to integrate bioactive membrane proteins has been demonstrated. We have also synthesized lipid bolaamphiphiles, which are comprised of two polar head groups separated by one or two hydrophobic spacer groups as an alternate approach for fabricating membrane-mimetic materials comprised of membrane-spanning constituents.

MembraneIn serving as mobile reservoirs for enzymatically active transmembrane proteins, we believe that properties can be incorporated into thin films that may be able to enhance the performance of a variety of artificial organs, cell transplants, and biosensor systems. For example, the arterial endothelium displays anticoagulant activity principally through the expression of membrane-bound thrombomodulin (TM) and heparan sulfate (HS). Membrane–mimetic thin films containing thrombomodulin (TM) and/or heparin have been produced and their capacity to inhibit thrombin generation evaluated in a continuous flow system. Surface bound TM and heparin nearly abolished steady-state thrombin responses produced by tissue factor. Our research group has a number of projects focused on the design of anti-thrombogenic surfaces, as well as the development and application of computational models of surface-induced thrombosis.

Marra KG, Winger TM, Hanson SR, Chaikof EL. Cytomimetic biomaterials. 1. In-situ polymerization of phospholipids on an alkylated surface. Macromolecules 1997; 30:6483-6487.

Faucher KM, Sun XL, Chaikof EL. Fabrication and characterization of glycocalyx-mimetic surfaces. Langmuir 2003; 19:1664-1670.

Sun XL, Stabler CA, Cazalis CS, Chaikof EL. Carbohydrate and protein immobilization onto solid surfaces by sequential Diels-Alder and azide-alkyne cycloadditions. Bioconjugate Chemistry 2006; 17:52-57.

Tseng PY, Rele S, Sun, X-L, Chaikof EL. Membrane–mimetic films containing thrombomodulin and heparin inhibit tissue factor-induced thrombin generation in a flow model. Biomaterials 2006; 27:2637–2650.

Faucher KM, Wannant S, Caves J, Sun X-L, Apkarian RP, Chaikof EL. Fabrication of a phospholipid membrane-mimetic film on the luminal surface of an ePTFE vascular graft. Biomaterials 2006; 27:3473-3481.

Krishnamurthy VR, Wilson JT, Cui W, Song XZ, Lasanajak Y, Cummings RD, Chaikof EL. Chemoselective immobilization of peptides on abiotic and cell surfaces at controlled densities. Langmuir 2010; 26: 7675–7678.

Jordan SW, Chaikof EL. Simulated surface-induced thrombin generation in a flow field. Biophys J 2011; 101:276-286

Qu Z, Muthukrishnan S, Urlam MK, Haller CA, Jordan SW, Kumar VA, Marzec UM, Elkasabi Y, Lahann J, Hanson SR, Chaikof EL. A biologically active surface enzyme assembly that attenuates thrombus formation. Advanced Functional Materials 2011; 21: 4736–4743.

Soft Tissue Repair

patch-scaffoldAlthough the repair of the abdominal wall is among the most common class of operations performed by general surgeons in the US, results remain far from ideal due to chronic inflammation, poor integration of implanted biomaterials, and infection. We have ongoing efforts to develop biomaterials that serve as artificial fascia for the repair of abdominal wall hernias. These materials are generated from matrix protein analogues and do not contain residual antigenic and inflammatory factors found in donor animal and cadaver tissue products. Currently, we are investigating the addition of various bioactive constituents, as well as the application of fabrication techniques to enhance our control over the patch microstructure. Our objectives are optimal integration with host tissue, rapid development of neovasculature, cellularity within and around the patch, and durability in the presence of bacterial infection.

scaffold-image-strip

Sallach RE, Cui W, Wen J, Martinez A, Conticello VP, Chaikof EL. Elastin-mimetic protein polymers capable of physical and chemical crosslinking. Biomaterials 2009; 30: 409-422.

Sallach RE, Cui W, Balderrama F, Martinez AW, Wen J, Haller CA, Taylor JV, Wright ER, Long RC, Chaikof EL. Long-term biostability of self-assembling protein polymers in the absence of covalent crosslinking. Biomaterials 2010;31:779-91

Caves JM, Cui W, Wen J, Kumar VA, Haller CA, Chaikof EL. Elastin-like protein matrix reinforced with collagen microfibers for soft tissue repair. Biomaterials 2011; 32: 5371-5379.

Cell Transplantation

IsletsWhole organ pancreatic allografts using current immunosuppresive protocols have an expected graft survival as high as 86% at one year and 74% at 5 years after transplantation. Despite these encouraging results, the risk of major perioperative morbidity, the associated complications of chronic immunosuppresion therapy, and the persistent shortage of donor organ tissue remain limitations of this approach. As a consequence, pancreas transplantation continues to have a limited role in the management of diabetes. As an alternative approach, islet transplantation offers several important advantages over whole organ transplantation.

Cell_1First, islets can be maintained and manipulated more easily than whole organ grafts and may be harvested from donor grafts that otherwise would not be suitable for whole organ transplantation. Second, islet transplantation, in comparison to whole organ grafting, is associated with a considerable reduction in morbidity and mortality, a decrease in intensive care unit utilization, shorter hospital stays, with the promise of achieving major reductions in overall healthcare costs. Significant limitations remain in islet transplantation that can be addressed by innovative strategies at the interface of biology, bioengineering, and biomolecular engineering.

Cell_2A major obstacle in islet transplantation is the high rate of primary non-function and early islet destruction, which has been observed after intraportal islet infusion, both in animal models and in clinical trials. Substantial evidence now suggests that an acute blood mediated inflammatory injury is largely responsible for the observed functional stunning or destruction of islets and may well amplify subsequent immune reactions. We postulate that molecularly engineered systems, including conformal barriers, anti-thrombotic fusion proteins, and heparan sulfate glycomimetics that limit inflammatory and pro-coagulant responses at the graft-host interface will reduce early islet destruction and, thereby, enhance islet engraftment. In dampening the early innate inflammatory response, a concomitant improvement in long-term islet survival may be attained by modulating the evolution of an adaptive immune response.

Stabler CS, Sun XL, Chaikof EL. Surface re-engineering of pancreatic islets with recombinant azido-thrombomodulin. Bioconjugate Chemistry 2007; 18:1713-1715

Wilson J, Cui W, Chaikof EL. Layer-by-layer assembly of a conformal nano-thin PEO coating for intraportal islet transplantation. Nano Letters 2008; 8:1940-48.

Cui W, Wilson JT, Wen J, Angsana J, Qu Z, Stabler C, Haller CA, Chaikof EL. Thrombomodulin improves early outcomes after intraportal islet transplantation. Am J Transplantation 2009; 9:1308-1316.

Wilson JT, Krishnamurthy VR, Qu Z, Cui W, Chaikof EL. Non-covalent cell surface engineering with cationic graft copolymers. J Am Chem Soc 2009; 131:18228–18229

Wilson JT, Haller CA, Qu Z, Cui W, Urlam MK, Chaikof EL. Biomolecular surface engineering of pancreatic islets with thrombomodulin. Acta Biomaterialia 2010; 6:1895-1903

Cui W, Angsana J, Wen J, Chaikof EL. Liposomal formulations of thrombomodulin increase engraftment after intraportal islet transplantation. Cell Transplantation 2010; 19:1359-1367.

Wilson JT, Cui W, Kozlovskaya V, Kharlampieva E, Pan D, Qu Z, Krishnamurthy VR, Mets J, Kumar V, Wen J, Song Y, Tsukruk VV, Chaikof EL. Cell surface engineering with polyelectrolyte multilayer thin films. J Am Chem Soc 2011; 133:7054-64.

Controlling Vascular Wall Healing

Healing_1The treatment of lower extremity vascular disease through the use of balloon or laser angioplasty, stenting, or atherectomy remains limited by a significant incidence of restenosis. Thus, new approaches that target thrombotic and inflammatory events, which contribute to restenosis and delayed vessel wall healing are required. We postulate that thrombin and purinergic dependant pathways can be inhibited by antibody mediated targeting of thrombomodulin, CD39, and CD73 to the site of vessel wall injury. Moreover, we hypothesize that nanoparticles produced from recombinant protein polymers will provide an effective mechanism for site-specific delivery of resolvins and their metabolic precursors that will further accelerate both the resolution of the inflammatory response and the reconstitution a functionally intact endothelium. As such, we anticipate that by abrogating early inflammatory and thrombotic responses, intimal hyperplasia will be limited after catheter-based interventions.

Healing_2   Healing_4   Healing_3

Kim W, Thévenot J, Ibarboure E, Lecommandoux S, Chaikof EL. Self-assembly of thermally responsive amphiphilic diblock copolypeptides into spherical micellar nanoparticles. Angew Chemie – International 2010; 49:4257-4260.

Krishnamurthy VR, Dougherty A, Haller CA, Chaikof EL. Total synthesis and bioactivity of 18R-hydroxy eicosapentaenoic acid. J Organic Chemistry 2011; 76: 5433-5437

Topcic D, Kim W, Holien JK, Jia F, Armstrong PC, Hohmann JD, Straub A, Krippner G, Haller CA, Domeij H, Hagemeyer CE, Parker MW, Chaikof EL*, Peter KP*. An activation-specific platelet inhibitor that can be turned on/off by medically employed hypothermia. Arteriosclerosis Thromb Vasc Biol 2011; 31:2015-23.

Kim W, Xiao J, Chaikof EL. Recombinant amphiphilic protein micelles for drug delivery. Langmuir 2011; 27: 14329-14334

Kim W, Brady C, Chaikof EL. Amphiphilic protein micelles for targeted in vivo imaging. In press Acta Biomaterialia 2012; 8:2476-2482.

Preventing and Treating Aortic Aneurysms

The cause of an aortic aneurysm remains poorly understood and successful pharmacotherapy is lacking despite the role of aneurysms as a major source of morbidity and death. Unregulated inflammatory and tissue repair processes underlie the maladaptive response of the vascular wall that leads to aneurysm formation.

AAA_1   AAA_2   AAA_3

Thus, there is great motivation for understanding the interplay between the complex biochemical, cellular, and biomechanical phenomena, which control inflammation and tissue repair in the vascular wall. We postulate that expression and shedding of the heparan sulfate proteoglycan syndecan-1 may provide an importantmechanism that inhibits abdominal aortic aneurysm (AAA) formation by limiting proteolytic or inflammatory activity and by promoting local reparative responses. The experimental approach within this project area is designed to yield fundamental knowledge regarding the regulated expression and shedding of syndecan-1, which we believe is an important molecular determinant modulating inflammatory and tissue repair processes in the vascular wall.

Li L, Chaikof EL. Mechanical stress regulates syndecan-4 expression and redistribution in vascular smooth muscle cells. Arteriosclerosis Thromb Vasc Biol 2002; 22:61-68.

Houston M, Julien MA, Parthasarathy S, Chaikof EL. Oxidized linoleic acid regulates expression and shedding of syndecan 4. Am J Physiol – Cell Physiol. 2005; 288:C458-66.

Julien MA, Haller CA, Wang P, Wen J, Chaikof EL. Mechanical strain induces a persistent upregulation of syndecan-1 expression in smooth muscle cells. J Cell Physiology 2007; 211:167-173.

Xiao J, Angsana J, Wen J, Smith SV, Park PW, Ford ML, Haller CA, Chaikof EL. Syndecan-1 displays a protective role in aortic aneurysm formation by modulating T cell-mediated responses. Arteriosclerosis Thromb Vasc Biol 2012; 32:386-96.