Glenn D. Prestwich

Hyaluronic Acid Hydrogels for Biomedical Applications

Hyaluronic acid (HA), an immunoneutral polysaccharide that is ubiquitous in the human body, is crucial for many cellular and tissue functions and has been in clinical use for over thirty years. When chemically modified, HA can be transformed into many physical forms-viscoelastic solutions, soft or stiff hydrogels, electrospun fibers, non-woven meshes, macroporous and fibrillar sponges, flexible sheets, and nanoparticulate fluids-for use in a range of preclinical and clinical settings. Many of these forms are derived from the chemical crosslinking of pendant reactive groups by addition/condensation chemistry or by radical polymerization. Clinical products for cell therapy and regenerative medicine require crosslinking chemistry that is compatible with the encapsulation of cells and injection into tissues. Moreover, an injectable clinical biomaterial must meet marketing, regulatory, and financial constraints to provide affordable products that can be approved, deployed to the clinic, and used by physicians. Many HA-derived hydrogels meet these criteria, and can deliver cells and therapeutic agents for tissue repair and regeneration. This progress report covers both basic concepts and recent advances in the development of HA-based hydrogels for biomedical applications.

Identification of an intracellular receptor for lysophosphatidic acid (LPA): LPA is a transcellular PPARγ agonist

Lysophosphatidic acid (LPA) is a pluripotent lipid mediator acting through plasma membrane-associated LPAx receptors that transduce many, but not all, of its effects. We identify peroxisome proliferator-activated receptor γ (PPARγ) as an intracellular receptor for LPA. The transcription factor PPARγ is activated by several lipid ligands, but agonists derived from physiologic signaling pathways are unknown. We show that LPA, but not its precursor phosphatidic acid, displaces the drug rosiglitazone from the ligand-binding pocket of PPARγ. LPA and novel LPA analogs we made stimulated expression of a PPAR-responsive element reporter and the endogenous PPARγ-controlled gene CD36, and induced monocyte lipid accumulation from oxidized low-density lipoprotein via the CD36 scavenger receptor. The synthetic LPA analogs were effective PPARγ agonists, but were poor ones for LPA1, LPA2, or LPA3 receptor transfected cells. Transfection studies in yeast, which lack nuclear hormone and LPAx receptors, show that LPA directly activates PPARγ. A major growth factor of serum is LPA generated by thrombin-activated platelets, and media from activated platelets stimulated PPARγ function in transfected RAW264.7 macrophages. This function was suppressed by ectopic LPA-acyltransferase expression. LPA is a physiologic PPARγ ligand, placing PPARγ in a signaling pathway, and PPARγ is the first intracellular receptor identified for LPA. Moreover, LPA produced by stimulated plasma platelets activates PPARγ in nucleated cells.

Characterization of Two Alternately Spliced Forms of Phospholipase D1 ACTIVATION OF THE PURIFIED ENZYMES BY PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE, ADP-RIBOSYLATION FACTOR, AND RHO FAMILY MONOMERIC GTP-BINDING PROTEINS AND PROTEIN KINASE C-α

We previously reported the cloning of a cDNA encoding human phosphatidylcholine-specific phospholipase D1 (PLD1), an ADP-ribosylation factor (ARF)-activated phosphatidylcholine-specific phospholipase D (Hammond, S. M., Tsung, S., Autschuller, Y., Rudge, S. A., Rose, K., Engebrecht, J., Morris, A. J., and Frohman, M. A. (1995) J. Biol. Chem. 270, 29640-29643). We have now identified an evolutionarily conserved shorter splice variant of PLD1 lacking 38 amino acids (residues 585-624) that arises from regulated splicing of an alternate exon. Both forms of PLD1 (PLD1a and 1b) have been expressed in Sf9 cells using baculovirus vectors and purified to homogeneity by detergent extraction and immunoaffinity chromatography. PLD1a and 1b have very similar properties. PLD1a and 1b activity is Mg2+dependent but insensitive to changes in free Ca2+ concentration. Phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate activate PLD1a and 1b but a range of other acidic phospholipids are ineffective. PLD1a and 1b are highly responsive to activation by GTP-γS-liganded ADP-ribosylation factor-1 (ARF-1) and can also be activated to a lesser extent by three purified RHO family monomeric GTP-binding proteins, RHO A, RAC-1, and CDC42. Activation of PLD1a and 1b by the RHO family monomeric GTP-binding proteins is GTP-dependent and synergistic with ARF-1. Purified protein kinase C-α activates PLD1a and 1b in a manner that is stimulated by phorbol esters and does not require ATP. Activation of PLD1a and 1b by protein kinase C-α is synergistic with ARF and with the RHO family monomeric GTP-binding proteins, suggesting that these three classes of regulators interact with different sites on the enzyme.

A PtdInsP(3)- and Rho GTPase-mediated positive feedback loop regulates neutrophil polarity.

When presented with a gradient of chemoattractant, many eukaryotic cells respond with polarized accumulation of the phospholipid PtdIns(3,4,5)P3. This lipid asymmetry is one of the earliest readouts of polarity in neutrophils, Dictyostelium discoideum and fibroblasts. However, the mechanisms that regulate PtdInsP3 polarization are not well understood. Using a cationic lipid shuttling system, we have delivered exogenous PtdInsP3 to neutrophils. Exogenous PtdInsP3 elicits accumulation of endogenous PtdInsP3 in a positive feedback loop that requires endogenous phosphatidylinositol-3-OH kinases (PI(3)Ks) and Rho family GTPases. This feedback loop is important for establishing PtdInsP3 polarity in response to both chemoattractant and to exogenous PtdInsP3; it may function through a self-organizing pattern formation system. Emergent properties of positive and negative regulatory links between PtdInsP3 and Rho family GTPases may constitute a broadly conserved module for the establishment of cell polarity during eukaryotic chemotaxis.

Discovering modes of action for therapeutic compounds using a genome-wide screen of yeast heterozygotes.

Modern medicine faces the challenge of developing safer and more effective therapies to treat human diseases. Many drugs currently in use were discovered without knowledge of their underlying molecular mechanisms. Understanding their biological targets and modes of action will be essential to design improved second-generation compounds. Here, we describe the use of a genome-wide pool of tagged heterozygotes to assess the cellular effects of 78 compounds in Saccharomyces cerevisiae. Specifically, lanosterol synthase in the sterol biosynthetic pathway was identified as a target of the antianginal drug molsidomine, which may explain its cholesterol-lowering effects. Further, the rRNA processing exosome was identified as a potential target of the cell growth inhibitor 5-fluorouracil. This genome-wide screen validated previously characterized targets or helped identify potentially new modes of action for over half of the compounds tested, providing proof of this principle for analyzing the modes of action of clinically relevant compounds.

Hyaluronan-binding proteins : tying up the giant

The ubiquitous glycosaminoglycan (GAG) hyaluronan has diverse biological roles in vertebrates. These include acting as a vital structural component of connective tissues, the formation of loose hydrated matrices that allow cells to divide and migrate (e.g. during development), immune cell adhesion and activation, and a role in intracellular signaling (1–3). This wide range of activities may seem surprising for an unbranched polysaccharide comprised entirely of a repeating disaccharide, D-glucuronic acid( 133)N-acetyl-D-glucosamine( 134), which (unlike other GAGs) is neither attached to a protein core nor Oor N-sulfated. Such diversity results in fact from the large number of hyaluronan-binding proteins (often termed hyaladherins) that exhibit significant differences in their tissue expression, cellular localization, specificity, affinity, and regulation. Therefore, characterization of the molecular basis of hyaluronan recognition by proteins and how this is modulated in vivo is an important key to understanding the biology of this GAG. In this article, we review the structural organization of vertebrate hyaladherins and how this may contribute to their different biological activities.

Cross-linked hyaluronic acid hydrogel films: new biomaterials for drug delivery.

A new hyaluronic acid (HA)-based hydrogel film was prepared and evaluated for use in drug delivery. This biocompatible material crosslinks and gels in minutes, and the dried film swells and rehydrates to a flexible hydrogel in seconds. HA was first converted to the adipic dihydrazide derivative and then crosslinked with the macromolecular homobifunctional reagent poly(ethylene glycol)-propiondialdehyde to give a polymer network. After gelation, a solvent casting method was used to obtain a HA hydrogel film. The dried film swelled sevenfold in volume in buffer, reaching equilibrium in less than 100 s. Scanning electron microscopy (SEM) of the hydrogel films showed a condensed and featureless structure before swelling, but a porous microstructure when hydrated. The thermal behavior of the hydrogel films was characterized by differential scanning calorimetry. The enzymatic degradation of the HA hydrogel films by hyaluronidase was studied using both SEM and a spectrophotometric assay. Drug release from the hydrogel film was evaluated in vitro using selected anti-bacterial and anti-inflammatory drugs. This novel biomaterial can be employed for controlled release of therapeutic agents at wound sites.

The PHD Finger of the Chromatin-Associated Protein ING2 Functions as a Nuclear Phosphoinositide Receptor

Phosphoinositides (PtdInsPs) play critical roles in cytoplasmic signal transduction pathways. However, their functions in the nucleus are unclear, as specific nuclear receptors for PtdInsPs have not been identified. Here, we show that ING2, a candidate tumor suppressor protein, is a nuclear PtdInsP receptor. ING2 contains a plant homeodomain (PHD) finger, a motif common to many chromatin-regulatory proteins. We find that the PHD fingers of ING2 and other diverse nuclear proteins bind in vitro to PtdInsPs, including the rare PtdInsP species, phosphatidylinositol 5-phosphate (PtdIns(5)P). Further, we demonstrate that the ING2 PHD finger interacts with PtdIns(5)P in vivo and provide evidence that this interaction regulates the ability of ING2 to activate p53 and p53-dependent apoptotic pathways. Together, our data identify the PHD finger as a phosphoinositide binding module and a nuclear PtdInsP receptor, and suggest that PHD-phosphoinositide interactions directly regulate nuclear responses to DNA damage.

Controlled chemical modification of hyaluronic acid: synthesis, applications, and biodegradation of hydrazide derivatives.

Controlled modification of the carboxylic acid moieties of hyaluronic acid with mono- and polyfunctional hydrazides leads to biochemical probes, biopolymers with altered physical and chemical properties, tethered drugs for controlled release, and crosslinked hydrogels as biocompatible scaffoldings for tissue engineering. Methods for polyhydrazide synthesis, for prodrug preparation, for hydrogel crosslinking, and for monitoring biodegradation are described.

Bioprinting vessel-like constructs using hyaluronan hydrogels crosslinked with tetrahedral polyethylene glycol tetracrylates.

Bioprinting enables deposition of cells and biomaterials into spatial orientations and complexities that mirror physiologically relevant geometries. To facilitate the development of bioartificial vessel-like grafts, two four-armed polyethylene glycol (PEG) derivatives with different PEG chain lengths, TetraPEG8 and TetraPEG13, were synthesized from tetrahedral pentaerythritol derivatives. The TetraPEGs are unique multi-armed PEGs with a compact and symmetrical core. The TetraPEGs were converted to tetra-acrylate derivatives (TetraPAcs) which were used in turn to co-crosslink thiolated hyaluronic acid and gelatin derivatives into extrudable hydrogels for printing tissue constructs. First, the hydrogels produced by TetraPAc crosslinking showed significantly higher shear storage moduli when compared to PEG diacrylate (PEGDA)-crosslinked synthetic extracellular matrices (sECMs) of similar composition. These stiffer hydrogels have rheological properties more suited to bioprinting high-density cell suspensions. Second, TetraPAc-crosslinked sECMs were equivalent or superior to PEGDA-crosslinked gels in supporting cell growth and proliferation. Third, the TetraPac sECMs were employed in a proof-of-concept experiment by encapsulation of NIH 3T3 cells in sausage-like hydrogel macrofilaments. These macrofilaments were then printed into tubular tissue constructs by layer-by-layer deposition using the Fab@Home printing system. LIVE/DEAD viability/cytotoxicity-stained cross-sectional images showed the bioprinted cell structures to be viable in culture for up to 4 weeks with little evidence of cell death. Thus, biofabrication of cell suspensions in TetraPAc sECMs demonstrates the feasibility of building bioartificial blood vessel-like constructs for research and potentially clinical uses.