Synthesis and Optimization of Collagen-targeting Peptide-Glycosaminoglycans for Inhibition of Platelets Following Endothelial Injury.

Many endothelial complications, whether from surgical or pathological origins, can result in the denudation of the endothelial layer and the exposure of collagen. Exposure of collagen results in the activation of platelets, leading to thrombotic and inflammatory cascades that ultimately result in vessel stenosis. We have previously reported the use of peptide-GAG compounds to target exposed collagen following endothelial injury. In this paper we optimize the spacer sequence of our collagen binding peptide to increase its conjugation to GAG backbones and increase the peptide-GAG collagen binding affinity by increasing peptide C-terminal cationic charge. Furthermore, we demonstrate the use of these molecules to inhibit platelet activation through collagen blocking, as well as their localization to exposed vascular collagen following systemic delivery. Altogether, optimization of peptide sequence and linkage chemistry can allow for increased conjugation and function, having implications for glycoconjugate use in other clinical applications.

Incorporation of a Collagen-Binding Chondroitin Sulfate Molecule to a Collagen Type I and II Blend Hydrogel for Cartilage Tissue Engineering.

Adding chondroitin sulfate (CS) to collagen scaffolds has been shown to improve the outcomes for articular cartilage tissue engineering. Instead of physical entrapment or chemical crosslinking of CS within a scaffold, this study investigated the use of CS with attached collagen-binding peptides (termed CS-SILY). This method better recapitulates the aspects of native cartilage while retaining CS within a collagen type I and II blend (Col I/II) hydrogel. CS retention, average fibril diameter, and mechanical properties were altered by varying the number of SILY peptides attached to the CS backbone. When mesenchymal stromal cells (MSCs) were encapsulated within the scaffolds, the addition of CS-SILY molecules resulted in higher sulfated glycosaminoglycan production, and these results suggest that CS-SILY promotes MSC differentiation into chondrocytes. Taken together, our study shows the promise of adding a CS-SILY molecule to a Col I/II hydrogel with encapsulated MSCs to promote cartilage repair.

Bioactive extracellular matrix scaffolds engineered with proangiogenic proteoglycan mimetics and loaded with endothelial progenitor cells promote neovascularization and diabetic wound healing.

Diabetic ischemic wound treatment remains a critical clinical challenge. Neovascularization plays a significant role in wound healing during all stages of the tissue repair process. Strategies that enhance angiogenesis and neovascularization and improve ischemic pathology may promote the healing of poor wounds, particularly diabetic wounds in highly ischemic conditions. We previously identified a cyclic peptide LXW7 that specifically binds to integrin αvß3 on endothelial progenitor cells (EPCs) and endothelial cells (ECs), activates vascular endothelial growth factor (VEGF) receptors, and promotes EC growth and maturation. In this study, we designed and synthesized a multi-functional pro-angiogenic molecule by grafting LXW7 and collagen-binding peptides (SILY) to a dermatan sulfate (DS) glycosaminoglycan backbone, named LXW7-DS-SILY, and further employed this multi-functional molecule to functionalize collagen-based extracellular matrix (ECM) scaffolds. We confirmed that LXW7-DS-SILY modification significantly promoted EPC attachment and growth on the ECM scaffolds in vitro and supported EPC survival in vivo in the ischemic environment. When applied in an established Zucker Diabetic Fatty (ZDF) rat ischemic skin flap model, LXW7-DS-SILY-functionalized ECM scaffolds loaded with EPCs significantly improved wound healing, enhanced neovascularization and modulated collagen fibrillogenesis in the ischemic environment. Altogether, this study provides a promising novel treatment to accelerate diabetic ischemic wound healing, thereby reducing limb amputation and mortality of diabetic patients.

Proangiogenic Collagen-Binding Glycan Therapeutic Promotes Endothelial Cell Angiogenesis.

Stimulating angiogenesis during wound healing continues to present a significant clinical challenge, given the limitations of current strategies to maintain therapeutic doses of growth factors and endothelial cell efficacy. Incorporating a balance of specific cues to encourage endothelial cell engraftment and cytokines to facilitate angiogenesis is necessary for blood vessel growth in the proinflammatory wound environment. Here, we incorporate a previously designed peptide (LXW7) capable of binding to the αvß3 integrin of endothelial cells with a dermatan sulfate glycosaminoglycan backbone grafted with collagen-binding peptides (SILY). By exploiting αvß3 integrin-mediated VEGF signaling, we propose an alternative strategy to overcome shortcomings of traditional growth factor therapy while homing the peptide to the wound bed. In this study, we describe the synthesis and optimization of LXW7-DS-SILY (LDS) variants and evaluate their angiogenic potential in vitro and in vivo. LDS displayed binding to collagen and endothelial cells. In vitro, the LDS variant with six LXW7 peptides increased endothelial cell proliferation, migration, and tubule formation through increased VEGFR2 phosphorylation compared to nontreated controls. In an in vivo chick chorioallantoic membrane assay, LDS laden collagen hydrogels increased blood vessel formation by 43% in comparison to the organism matched blank hydrogels. Overall, these findings demonstrate the potential of a robust targeted glycan therapeutic for promoting angiogenesis during wound healing.

Localized inhibition of platelets and platelet derived growth factor by a matrix targeted glycan mimetic significantly attenuates liver fibrosis.

New therapeutic strategies are needed for the growing unmet clinical needs in liver disease and fibrosis. Platelet activation and PDGF activity are recognized as important therapeutic targets; however, no therapeutic approach has yet addressed these two upstream drivers of liver fibrosis. We therefore designed a matrix-targeting glycan therapeutic, SBR-294, to inhibit collagen-mediated platelet activation while also inhibiting PDGF activity. Herein we describe the synthesis and characterization of SBR-294 and demonstrate its potential therapeutic benefits in vitro and in vivo. In vitro SBR-294 was found to bind collagen (EC50 = 23 nM), thereby inhibiting platelet-collagen engagement (IC50 = 60 nM). Additionally, SBR-294 was found to bind all PDGF homodimeric isoforms and to inhibit PDGF-BB mediated hepatic stellate cell activation and proliferation. Translating these mechanisms in vivo, SBR-294 reduced fibrosis by up to 54% in the CCl4 mouse model (p = 0.0004), as measured by Sirius red histological analysis. Additional fibrosis measurements were also supportive of the therapeutic benefit in this model. These results support the therapeutic benefit of platelet and PDGF antagonism and warrant further investigation of SBR-294 as a potential treatment for liver fibrosis.

Best of Both Hydrogel Worlds: Harnessing Bioactivity and Tunability by Incorporating Glycosaminoglycans in Collagen Hydrogels.

Collagen, the most abundant protein in mammals, has garnered the interest of scientists for over 50 years. Its ubiquitous presence in all body tissues combined with its excellent biocompatibility has led scientists to study its potential as a biomaterial for a wide variety of biomedical applications with a high degree of success and widespread clinical approval. More recently, in order to increase their tunability and applicability, collagen hydrogels have frequently been co-polymerized with other natural and synthetic polymers. Of special significance is the use of bioactive glycosaminoglycans-the carbohydrate-rich polymers of the ECM responsible for regulating tissue homeostasis and cell signaling. This review covers the recent advances in the development of collagen-based hydrogels and collagen-glycosaminoglycan blend hydrogels for biomedical research. We discuss the formulations and shortcomings of using collagen in isolation, and the advantages of incorporating glycosaminoglycans (GAGs) in the hydrogels. We further elaborate on modifications used on these biopolymers for tunability and discuss tissue specific applications. The information presented herein will demonstrate the versatility and highly translational value of using collagen blended with GAGs as hydrogels for biomedical engineering applications.

PEPTIDE-MODIFIED CHONDROITIN SULFATE REDUCES COEFFICIENT OF FRICTION AT ARTICULAR CARTILAGE SURFACE.

Osteoarthritis is a debilitating disease that results in pain and joint stiffness. Currently, steroidal and nonsteroidal anti-inflammatory drugs and supplements aimed at restoring lubrication to the affected joint are the most successful with respect to improving patient comfort. Due to the success in lubricating therapies, there exists a keen interest to develop better therapies that mimic how lubrication occurs naturally in the joint. Here we describe the results obtained using a chondroitin sulfate chain to which is conjugated peptides that bind to either hyaluronic acid (found in high concentrations in the synovial fluid) or collagen type II (present on the cartilage surface). Our study investigates the effect of binding to the cartilage surface and interacting with hyaluronic acid on lubrication at the cartilage surface. The results described here suggest that binding to the cartilage surface is critical to supporting lubrication and did not require the addition of hyaluronic acid to reduce friction.

Rapid endothelialization of small diameter vascular grafts by a bioactive integrin-binding ligand specifically targeting endothelial progenitor cells and endothelial cells.

Establishing and maintaining a healthy endothelium on vascular and intravascular devices is crucial for the prevention of thrombosis and stenosis. Generating a biofunctional surface on vascular devices to recruit endothelial progenitor cells (EPCs) and endothelial cells (ECs) has proven efficient in promoting in situ endothelialization. However, molecules conventionally used for EPC/EC capturing generally lack structural stability, capturing specificity, and biological functionalities, which have limited their applications. Discovery of effective, specific, and structurally stable EPC/EC capturing ligands is desperately needed. Using the high-throughput One-Bead One-Compound combinatorial library screening technology, we recently identified a disulfide cyclic octa-peptide LXW7 (cGRGDdvc), which possesses strong binding affinity and functionality to EPCs/ECs, weak binding to platelets, and no binding to inflammatory cells. Because LXW7 is cyclic and 4 out of the 8 amino acids are unnatural D-amino acids, LXW7 is highly proteolytically stable. In this study, we applied LXW7 to modify small diameter vascular grafts using a Click chemistry approach. In vitro studies demonstrated that LXW7-modified grafts significantly improved EPC attachment, proliferation and endothelial differentiation and suppressed platelet attachment. In a rat carotid artery bypass model, LXW7 modification of the small diameter vascular grafts significantly promoted EPC/EC recruitment and rapidly achieved endothelialization. At 6 weeks after implantation, LXW7-modified grafts retained a high patency of 83%, while the untreated grafts had a low patency of 17%. Our results demonstrate that LXW7 is a potent EPC/EC capturing and platelet suppressing ligand and LXW7-modified vascular grafts rapidly generate a healthy and stable endothelial interface between the graft surface and the circulation to reduce thrombosis and improve patency. STATEMENT OF SIGNIFICANCE: In this study, One-Bead One-Compound (OBOC) technology has been applied for the first time in discovering bioactive ligands for tissue regeneration applications. Current molecules used to modify artificial vascular grafts generally lack EPC/EC capturing specificity, biological functionalities and structural stability. Using OBOC technology, we identified LXW7, a constitutionally stable disulfide cyclic octa-peptide with strong binding affinity and biological functionality to EPCs/ECs, very weak binding to platelets and no binding to inflammatory cells. These characteristics are crucial for promoting rapid endothelialization to prevent thrombosis and improve patency of vascular grafts. LXW7 coating technology could be applied to a wide range of vascular and intravascular devices, including grafts, stents, cardiac valves, and catheters, where a "living" endothelium and healthy blood interface are needed.

Proteoglycans in Biomedicine: Resurgence of an Underexploited Class of ECM Molecules.

Proteoglycans have emerged as biomacromolecules with important roles in matrix remodeling, homeostasis, and signaling in the past two decades. Due to their negatively charged glycosaminoglycan chains as well as distinct core protein structures, they interact with a variety of molecules, including matrix proteins, growth factors, cytokines and chemokines, pathogens, and enzymes. This led to the dawn of glycan therapies in the 20th century, but this research was quickly overshadowed by readily available DNA and protein-based therapies. The recent development of recombinant technology and advances in our understanding of proteoglycan function have led to a resurgence of these molecules as potential therapeutics. This review focuses on the recent preclinical efforts that are bringing proteoglycan research and therapies back to the forefront. Examples of studies using proteoglycan cores and mimetics have also been included to give the readers a perspective on the wide-ranging and extensive applications of these versatile molecules. Collectively, these advances are opening new avenues for targeting diseases at a molecular level, and providing avenues for the development of new and exciting treatments in regenerative medicine.

Incorporation of types I and III collagen in tunable hyaluronan hydrogels for vocal fold tissue engineering.

Vocal fold scarring is the fibrotic manifestation of a variety of voice disorders, and is difficult to treat. Tissue engineering therapies provide a potential strategy to regenerate the native tissue microenvironment in order to restore vocal fold functionality. However, major challenges remain in capturing the complexity of the native tissue and sustaining regeneration. We hypothesized that hydrogels with tunable viscoelastic properties that present relevant biological cues to cells might be better suited as therapeutics. Herein, we characterized the response of human vocal fold fibroblasts to four different biomimetic hydrogels: thiolated hyaluronan (HA) crosslinked with poly(ethylene glycol) diacrylate (PEGDA), HA-PEGDA with type I collagen (HA-Col I), HA-PEGDA with type III collagen (HA-Col III) and HA-PEGDA with type I and III collagen (HA-Col I-Col III). Collagen incorporation allowed for interpenetrating fibrils of collagen within the non-fibrillar HA network, which increased the mechanical properties of the hydrogels. The addition of collagen fibrils also reduced hyaluronidase degradation of HA and hydrogel swelling ratio. Fibroblasts encapsulated in the HA-Col gels adopted a spindle shaped fibroblastic morphology by day 7 and exhibited extensive cytoskeletal networks by day 21, suggesting that the incorporation of collagen was essential for cell adhesion and spreading. Cells remained viable and synthesized new DNA throughout 21 days of culture. Gene expression levels significantly differed between the cells encapsulated in the different hydrogels. Relative fold changes in gene expression of MMP1, COL1A1, fibronectin and decorin suggest higher degrees of remodeling in HA-Col I-Col III gels in comparison to HA-Col I or HA-Col III hydrogels, suggesting that the former may better serve as a natural biomimetic hydrogel for tissue engineering applications. STATEMENT OF SIGNIFICANCE: Voice disorders affect about 1/3rd of the US population and significantly reduce quality of life. Patients with vocal fold fibrosis have few treatment options. Tissue engineering therapies provide a potential strategy to regenerate the native tissue microenvironment in order to restore vocal fold functionality. Various studies have used collagen or thiolated hyaluronan (HA) with gelatin as potential tissue engineering therapies. However, there is room for improvement in providing cells with more relevant biological cues that mimic the native tissue microenvironment and sustain regeneration. The present study introduces the use of type I collagen and type III collagen along with thiolated HA as a natural biomimetic hydrogel for vocal fold tissue engineering applications.

An in vitro scaffold-free epithelial-fibroblast coculture model for the larynx.

OBJECTIVES/HYPOTHESIS: Physiologically relevant, well-characterized in vitro vocal fold coculture models are needed to test the effects of various challenges and therapeutics on vocal fold physiology. We characterize a healthy state coculture model, created by using bronchial/tracheal epithelial cells and immortalized vocal fold fibroblasts. We also demonstrate that this model can be induced into a fibroplastic state to overexpress stress fibers using TGFß1. STUDY DESIGN: In vitro. METHODS: Cell metabolic activity of immortalized human vocal fold fibroblasts incubated in different medium combinations was confirmed with an MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay. Fibroblasts were grown to confluence, and primary bronchial/tracheal epithelial cells suspended in coculture medium were seeded directly over the base layer of the fibroblasts. Cells were treated with transforming growth factor ß1 (TGFß1) to induce myofibroblast formation. Cell shape and position were confirmed by live cell tracking, fibrosis was confirmed by probing for α smooth muscle actin (αSMA), and phenotype was confirmed by immunostaining for vimentin and E-cadherin. RESULTS: Fibroblasts retain metabolic activity in coculture epithelial medium. Live cell imaging revealed a layer of epithelial cells atop fibroblasts. αSMA expression was enhanced in TGFß1-treated cells, confirming that both cell types maintained a healthy phenotype in coculture, and can be induced into overexpressing stress fibers. Vimentin and E-cadherin immunostaining show that cells retain phenotype in coculture. CONCLUSIONS: These data lay effective groundwork for a functional coculture model that retains the reproducibility necessary to serve as a viable diagnostic and therapeutic screening platform. LEVEL OF EVIDENCE: NA Laryngoscope, 127:E185-E192, 2017.

A Review of Hyaluronic Acid and Hyaluronic Acid-based Hydrogels for Vocal Fold Tissue Engineering.

Vocal fold scarring is a common cause of dysphonia. Current treatments involving vocal fold augmentation do not yield satisfactory outcomes in the long term. Tissue engineering and regenerative medicine offer an attractive treatment option for vocal fold scarring, with the aim to restore the native extracellular matrix microenvironment and biomechanical properties of the vocal folds by inhibiting progression of scarring and thus leading to restoration of normal vocal function. Hyaluronic acid is a bioactive glycosaminoglycan responsible for maintaining optimum viscoelastic properties of the vocal folds and hence is widely targeted in tissue engineering applications. This review covers advances in hyaluronic acid-based vocal fold tissue engineering and regeneration strategies.

Acute Nanoparticle Exposure to Vocal Folds: A Laboratory Study.

OBJECTIVES: Airway exposure to nanoparticles is common in occupational settings. Inhaled nanoparticles have toxic effects on respiratory tissue. Vocal folds are also at direct risk from inhaled nanoparticles. This study investigated the effects of single-walled carbon nanotubes (SWCNT), a type of nanoparticle, on vocal fold epithelium and fibroblasts. These cell types were selected for study as the epithelium is the outer layer of the vocal folds and fibroblasts are the most common cell type in connective tissue underlying the epithelium. METHODS: Native porcine vocal fold epithelium and cultured human vocal fold fibroblasts were exposed to SWCNTs (100 ng/mL) and control (no SWCNT) in vitro. Epithelial and fibroblast viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Epithelial barrier integrity was assessed with transepithelial resistance and sodium fluorescein permeability. Epithelial tight junctional protein occludin expression was measured with Western blot. Gene expressions of the fibroblast-specific protein 1 (FSP-1), α-smooth muscle actin (α-SMA), and collagen III (Col-III) were assessed using quantitative polymerase chain reaction. RESULTS: Transcriptional expression of genes encoding FSP-1 and Col-III was increased significantly following SWCNT exposure. There were no significant differences between control and SWCNT groups on any of the other measures. CONCLUSIONS: SWCNT exposure induces vocal fold fibroblasts to a fibrotic phenotype. These data help us understand vocal fold defense mechanisms and lay the groundwork for studying the physiological effects of nanoparticle exposure in vivo.