Joshua Z. Gasiorowski

Hydroporation as the mechanism of hydrodynamic delivery.

We have reported that a rapid tail vein injection of a large volume of plasmid DNA solution into a mouse results in high level of transgene expression in the liver. Gene transfer efficiency of this hydrodynamics-based procedure is determined by the combined effect of a large volume and high injection speed. Here, we show that the hydrodynamic injection induces a transient irregularity of heart function, a sharp increase in venous pressure, an enlargement of liver fenestrae, and enhancement of membrane permeability of the hepatocytes. At the cellular level, our results suggest that hepatic delivery by the hydrodynamic injection is accomplished by the generation of membrane pores in the hepatocytes.

Multi-component extracellular matrices based on peptide self-assembly

Extracellular matrices (ECMs) are challenging design targets for materials synthesis because they serve multiple biological roles, and they are composed of multiple molecular constituents. In addition, their composition and activities are dynamic and variable between tissues, and they are difficult to study mechanistically in physiological contexts. Nevertheless, the design of synthetic ECMs is a central consideration in applications such as regenerative medicine and 3D cell culture. In order to produce synthetic matrices having both multi-component construction and high levels of compositional definition, strategies based on molecular self-assembly are receiving increasing interest. These approaches are described in this tutorial review and compared with the structures and processes in native ECMs that serve as their inspiration.

Modulating adaptive immune responses to peptide self-assemblies.

Self-assembling peptides and peptide derivatives have received significant interest for several biomedical applications, including tissue engineering, wound healing, cell delivery, drug delivery, and vaccines. This class of materials has exhibited significant variability in immunogenicity, with many peptides eliciting no detectable antibody responses but others eliciting very strong responses without any supplemental adjuvants. Presently, strategies for either avoiding strong antibody responses or specifically inducing them are not well-developed, even though they are critical for the use of these materials both within tissue engineering and within immunotherapies. Here, we investigated the molecular determinants and immunological mechanisms leading to the significant immunogenicity of the self-assembling peptide OVA-Q11, which has been shown previously to elicit strong antibody responses in mice. We show that these responses can last for at least a year. Using adoptive transfer experiments and T cell knockout models, we found that these strong antibody responses were T cell-dependent, suggesting a route for avoiding or ensuring immunogenicity. Indeed, by deleting amino acid regions in the peptide recognized by T cells, immunogenicity could be significantly diminished. Immunogenicity could also be attenuated by mutating key residues in the self-assembling domain, thus preventing fibrillization. A second self-assembling peptide, KFE8, was also nonimmunogenic, but nanofibers of OVA-KFE8 elicited strong antibody responses similar to OVA-Q11, indicating that the adjuvant action was not dependent on the specific self-assembling peptide sequence. These findings will facilitate the design of self-assembled peptide biomaterials, both for applications where immunogenicity is undesirable and where it is advantageous.

Gradated assembly of multiple proteins into supramolecular nanomaterials

Biomaterials displaying precise ratios of different bioactive protein components are critical for applications ranging from vaccines to regenerative medicine, but their design is often hindered by limited choices and cross-reactivity of protein conjugation chemistries. Here, we describe a strategy for inducing multiple different expressed proteins of choice to assemble into nanofibers and gels with exceptional compositional control. The strategy employs novel “βTail” tags, which allow for good protein expression in bacteriological cultures, yet can be induced to co-assemble into nanomaterials when mixed with additional β-sheet fibrillizing peptides. Multiple different βTail fusion proteins could be inserted into peptide nanofibers alone or in combination at predictable, smoothly gradated concentrations, providing a simple yet versatile route to install precise combinations of proteins into nanomaterials. The technology is illustrated by achieving precisely targeted hues using mixtures of fluorescent proteins, by creating nanofibers bearing enzymatic activity, and by adjusting antigenic dominance in vaccines.

Mechanisms of nuclear transport and interventions

One of the more overlooked aspects of drug action and delivery is the exploitation of nucleocytoplasmic shuttling. Eukaryotic cells regulate many biological processes by the compartmentation of specific proteins into designated areas. Drugs that have a direct effect on a single protein must be able to localize to the same site as the protein and interact with one or more of its domains. Alternatively, a drug that effectively blocks the target protein from reaching its proper organelle can also inhibit the proteins function. Exploiting the selective movement of macromolecules across the nuclear envelope represents an exciting new area of drug development. This review aims to explain the basic nuclear import/export pathways while focusing on the known drugs that alter the regulation of nucleocytoplasmic trafficking.

Alterations in gene expression of human vascular endothelial cells associated with nanotopographic cues

Human cells in vivo are exposed to a topographically rich, 3-dimenisional environment which provides extracellular cues initiating a cascade of biochemical signals resulting in changes in cell behavior. One primary focus of our group is the development of biomimetic substrates with anisotropic nanoscale topography to elucidate the mechanisms by which physical surface cues are translated into biochemical signals. To investigate changes in gene expression as a result of nanotopographic cues, Human Umbilical Vein Endothelial Cells (HUVECs) were cultured on chemically identical flat and 400 nm pitch nanogrooved surfaces. After 12 h, RNA was harvested for an Affymetrix HG U133 Plus 2.0 gene array. Of over 47,000 possible gene probes, 3171 had at least a two-fold difference in expression between the control flat and 400 nm pitch. The gene ontology groups with the most significant increase in expression are involved in protein modification and maintenance, similar to cells upregulating chaperone and protein synthesis genes in response to physical stresses. The most significant decreases in expression were observed with cell cycle proteins, including cyclins and checkpoint proteins. Extracellular matrix proteins, including integrins, collagens, and laminins, are almost uniformly downregulated on the 400 nm pitch surfaces compared to control. The downregulation of one of these genes, integrin beta 1, was confirmed via quantitative PCR. Together, these gene array data, in addition to our studies of cell behavior on nanoscale surfaces, contribute to our understanding of the signaling pathways modulated by topographical surface cues.

Response of Human Trabecular Meshwork Cells to Topographic Cues on the Nanoscale Level

UNLABELLED purpose To determine how primary human trabecular meshwork (HTM) cells are influenced by their interaction with nanopatterned substrates. METHODS HTM cells from several individuals were grown on planar or anisotropically ordered nanopatterned surfaces. Microscopy was used to measure cellular elongation and alignment. Cells were also incubated with 10(-7) M dexamethasone for comparison to control cells. Quantitative PCR for myocilin and versican isoforms was performed in addition to Western blots of myocilin and alphaB-crystallin. RESULTS Cells on anisotropically ordered nanopatterned substrates aligned with the surface nanopatterns and displayed actin filaments that were parallel to the patterned ridges and grooves. The cells became more elongated on the nanogrooved surfaces compared with the planar control cells. Myocilin mRNA and protein levels increased when HTM cells were plated onto 400-nm pitch surfaces. With some HTM cells, myocilin increased to a greater extent when untreated cells were plated on nanosurfaces compared with the cells grown on planar surfaces with dexamethasone. The V0 and V1 isoforms of versican had increased expression on patterned surfaces. CONCLUSIONS Nanopatterned surfaces containing biomimetic length scale features clearly influenced cellular behavior of HTM cells. Increased mRNA and protein levels of myocilin were observed when cells were grown on 400-nm pitch surfaces, suggesting that the reduction of myocilin mRNA when cells are plated onto flat tissue culture plastic is an artifact of a nonphysiologic culture environment that lacks appropriate topographic cues.

Biological Properties of Trabecular Meshwork Cells

The molecular and physiological mechanisms that lead to the progression of glaucoma are poorly understood. Despite the fact that glaucoma afflicts millions of people worldwide, research on the disease is limited by the current animal models that do not translate well to human forms of the disease. However, recent advances in culturing and manipulating human trabecular meshwork cells may provide a means to elucidate some of the mechanisms that cause glaucoma. This review focuses on the properties of trabecular meshwork cells, from their characteristic expression profile in vivo to their responsiveness to biochemical and biophysical signals in vitro. Hopefully the study of cultured trabecular meshwork cells will provide a better understanding of glaucoma and lead to new, much needed therapies.

Biophysical Cues and Cell Behavior: The Big Impact of Little Things

The extracellular matrix is composed of a variety of proteins, polysaccharides, and glycosaminoglycans that self-assemble into a hierarchical order of nanometer- to micrometer-scale fibrils and fibers. The shapes, sizes, and elasticity present within this highly ordered meshwork regulate behaviors in most cell types. It has been well documented that cellular migration, proliferation, differentiation, and tissue development are all influenced by matrix geometries and compliance, but how these external biophysical cues are translated into activated intracellular signaling cascades remains poorly understood. Fortunately, technological improvements in artificial substrate fabrication have provided biologists with tools to test cellular interactions within controlled three-dimensional environments. Here, we review cellular responses to biophysical cues and discuss their clinical relevancy and application. We focus especially on integrative approaches that aim to first characterize the properties of specific extracellular matrices and then precisely fabricate biomimetic materials to elucidate how relevant cells respond to the individual biophysical cues present in their native tissues. Through these types of comprehensive studies, biologists have begun to understand and appreciate how exceedingly small features can have a significant impact on the regulation, development, and homeostasis of cells and tissues.

The influence of substrate topography on the migration of corneal epithelial wound borders

Currently available artificial corneas can develop post-implant complications including epithelial downgrowth, infection, and stromal melting. The likelihood of developing these disastrous complications could be minimized through improved formation and maintenance of a healthy epithelium covering the implant. We hypothesize that this epithelial formation may be enhanced through the incorporation of native corneal basement membrane biomimetic chemical and physical cues onto the surface of the keratoprosthesis. We fabricated hydrogel substrates molded with topographic features containing specific bio-ligands and developed an in vitro wound healing assay. In our experiments, the rate of corneal epithelial wound healing was significantly increased by 50% in hydrogel surfaces containing topographic features, compared to flat surfaces with the same chemical attributes. We determined that this increased healing is not due to enhanced proliferation or increased spreading of the epithelial cells, but to an increased active migration of the epithelial cells. These results show the potential benefit of restructuring and improving the surface of artificial corneas to enhance epithelial coverage and more rapidly restore the formation of a functional epithelium.