Chemoselective Chemistry: Engineering Materials and Cell Surfaces to Control Biological Interactions

The development of strategies to control the interface between biomolecules and a solid support is critical to a number of research areas, including drug discovery, tissue engineering, and gene microarray technology. In particular, tremendous effort has been extended toward interfacing material science with cell biology to conduct mechanistic cell adhesion, polarization, and migration studies. These investigations require the combined use of a model substrate that mimics the complex nature of the extracellular matrix and a synthetic chemical immobilization methodology to pattern biospecific, biomolecular cues for cellular recognition. Currently, self-assembled monolayers (SAMs) of alkanethiolates on gold represent the most well-studied and developed surface systems in biointerfacial science, enabling the design and implementation of complex, dynamic substrates for controlling biological interactions at the molecular level. This research is focused on employing chemoselective chemistry to engineer materials and cell surfaces for the control of biological interactions. Thus, smart biosurfaces and materials were manipulated to investigate peptide-cell, protein-carbohydrate, and lipid-cell interactions. A library of biomolecules was designed and synthesized to include chemoselective and bio-orthogonal functional groups, ketone and oxyamine. With this coupling methodology, biomaterials and cell surfaces were successfully engineered to examine a variety of cell behaviors, such as cell-biospecific ligand interactions, adhesion, polarization, migration, and cellular response to other cells. Chapter 1 provides an introduction to SAMs and a general discussion regarding the design and utility of dynamic SAM surfaces and for biological analyses. The use of SAMs on gold and indium tin oxide for cell adhesion studies is presented in Chapters 2 and 3, respectively. Chapter 4 demonstrates the development and application of a renewable carbohydrate microarray based on hydroquinone-terminated SAMs on gold. Hydroquinone was then incorporated with cell adhesive peptide, RGD, to survey selected carbohydrates and peptides for their combined effect on fibroblast adhesion, morphology, and migration; this data is discussed in Chapter 5. A cell-surface engineering strategy based on liposome delivery and membrane fusion to direct cell-cell contacts and generate 3D tissue-like structures is reported in Chapters 6 and 7. Finally, Chapter 8 describes my general conclusions and future research directions

Long-Lived Engineering of Glycans to Direct Stem Cell Fate

Glycans mediate many critical, long-term biological processes, such as stem cell differentiation. However, few methods are available for the sustained remodeling of cells with specific glycan structures. A new strategy that enables the long-lived presentation of defined glycosaminoglycans on cell surfaces using HaloTag proteins (HTPs) as anchors is reported. By controlling the sulfation patterns of heparan sulfate (HS) on pluripotent embryonic stem cell (ESC) membranes, it is demonstrated that specific glycans cause ESCs to undergo accelerated exit from self-renewal and differentiation into neuronal cell types. Thus, the stable display of glycans on HTP scaffolds provides a powerful, versatile means to direct key signaling events and biological outcomes such as stem cell fate

Directing Neuronal Signaling through Cell-Surface Glycan Engineering

The ability to tailor plasma membranes with specific glycans may enable the control of signaling events that are critical for proper development and function. We report a method to modify cell surfaces with specific sulfated chondroitin sulfate (CS) glycosaminoglycans using chemically modified liposomes. Neurons engineered to display CS-E-enriched polysaccharides exhibited increased activation of neurotrophin-mediated signaling pathways and enhanced axonal growth. This approach provides a facile, general route to tailor cell membranes with biologically active glycans and demonstrates the potential to direct important cellular events through cell-surface glycan engineering

Remote Control of Tissue Interactions via Engineered Photo-switchable Cell Surfaces

We report a general cell surface molecular engineering strategy via liposome fusion delivery to create a dual photo-active and bio-orthogonal cell surface for remote controlled spatial and temporal manipulation of microtissue assembly and disassembly. Cell surface tailoring of chemoselective functional groups was achieved by a liposome fusion delivery method and quantified by flow cytometry and characterized by a new cell surface lipid pull down mass spectrometry strategy. Dynamic co-culture spheroid tissue assembly in solution and co-culture tissue multilayer assembly on materials was demonstrated by an intercellular photo-oxime ligation that could be remotely cleaved and disassembled on demand. Spatial and temporal control of microtissue structures containing multiple cell types was demonstrated by the generation of patterned multilayers for controlling stem cell differentiation. Remote control of cell interactions via cell surface engineering that allows for real-time manipulation of tissue dynamics may provide tools with the scope to answer fundamental questions of cell communication and initiate new biotechnologies ranging from imaging probes to drug delivery vehicles to regenerative medicine, inexpensive bioreactor technology and tissue engineering therapies

Worldwide Distribution and Extracutaneous Manifestations of Henoch-Schönlein Purpura in Adults: Narrative Review

BackgroundHenoch-Schönlein purpura (HSP), a leukocytoclastic small vessel vasculitis, exhibits both cutaneous and systemic manifestations. While predominantly observed in childhood, it may manifest in adults with more pronounced systemic involvement. Furthermore, HSP is a global phenomenon showcasing epidemiological and systemic variances. ObjectiveThis study aims to scrutinize extracutaneous manifestations in adults with HSP, discerning distinctions according to geographical regions on a worldwide scale. MethodsA comprehensive search encompassing PubMed, Embase, Cochrane Library, and Web of Science was executed, covering papers published from January 1, 1970, to December 1, 2019. Keywords used included “Henoch-Schönlein purpura,” “henoch schonlein purpura+adult,” “IgA vasculitis+adult,” “HSP+adult,” and “IgAV.” A total of 995 publications were identified, from which 42 studies encompassing 4064 patients were selected, with a predominant focus on cases reported in Asia, Europe, and the Americas. ResultsAmong adults afflicted with HSP, European patients exhibited a higher propensity for male predominance (P<.001), gastrointestinal involvement (P<.001), and musculoskeletal complications (P<.001). Conversely, patients from the Americas were least likely to experience genitourinary involvement (P<.001). ConclusionsHSP demonstrates a variance in distribution and extracutaneous manifestations within distinct geographical boundaries. In the adult population, European patients exhibited a higher prevalence of male gender and gastrointestinal and musculoskeletal involvement. Asian patients were more predisposed to genitourinary involvement when compared to their American counterparts. The establishment of prospective studies using standardized reporting measures is imperative to validate the relationships unveiled in this investigation

In Situ Modulation of Cell Behavior via Smart Dual-Ligand Surfaces

Due to the highly complex nature of the extracellular matrix (ECM), the design and implementation of dynamic, stimuli-responsive surfaces that present well-defined ligands and serve as model ECM substrates have been of tremendous interest to biomaterials, biosensor, and cell biology communities. Such tools provide strategies for identifying specific ligand–receptor interactions that induce vital biological consequences. Herein, we report a novel dual-ligand-presenting surface methodology that modulates dynamic ECM properties to investigate various cell behaviors. Peptides PHSRN, cRGD, and KKKTTK, which mimic the cell- and heparan sulfate-binding domains of fibronectin, and carbohydrates Gal and Man were combined with cell adhesive RGD to survey possible synergistic or antagonist ligand effects on cell adhesion, spreading, growth, and migration. Soluble molecule and enzymatic inhibition assays were also performed, and the levels of focal adhesion kinase in cells subjected to different ligand combinations were quantified. A redox-responsive trigger was incorporated into this surface strategy to spontaneously release ligands in the presence of adhered cells, and cell spreading, growth, and migration responses were measured and compared. The identity and nature of the dual-ligand combination directly influenced cell behavior

In cells committed to terminal cell death, caspase-1 and -8 activity is increased with LL-37 treatment and reduced with GM-0111.

<p><b>A</b>. The percentage of total HNEpCs and J774.2 cells with active caspase-1 and 7-AAD (late cell death marker) is significantly increased with LL-37 treatment, and this is dose-dependently reversed with GM-0111 treatment. <b>B</b>. The percentage of total HNEpCs and J774.2 cells with active caspase-8 and 7-AAD is significantly increased with LL-37 treatment and dose-dependently decreased with GM-0111. ****P ≤ 0.0001, *P ≤ 0.05, ns (not significant) P > 0.05. The data represent the means ± SD (n = 2–5). Note the differences in y-axis scale (panel B y-axis maximum only 1.5%), adjusted to allow visual resolution but heights of bars are not comparable between panels. <b>C</b>. There is a statistically significant though minimal (<1%) increase in caspase-3 or -7 activity in HNEpCs also positive for 7-AAD after LL-37 treatment, and the effect is reversed with GM-0111. There is no significant change in caspase-3 or -7 activity with LL-37 or GM-0111 in J774.2 cells.</p

Diagram of the proposed mechanism of LL-37-induced cell death and protection from GM-0111.

<p>Nasal epithelial cells subjected to LL-37 demonstrate a pro-inflammatory response, characterized by increased ATP, IL-6, and -8 production and pyroptosis and/or necrosis via caspase-1 and -8 but not caspase-3 or -7 activation. These changes are prevented by GM-0111 treatment. IL-6 and -8 promote an inflammatory response <i>in vivo</i> through the recruitment of neutrophils and further inflammatory signaling in a positive feedback loop. The process of pro-inflammatory cell death is propagated to nearby cells due to these changes in the local environment, resulting in an unchecked cytotoxic response initiated by LL-37.</p

LL-37 increases cell death, which is reduced by GM-0111.

<p><b>A</b>. Treatment with LL-37 alone results in a dose-dependent increase in late cell death (Annexin V⁺/7-AAD⁺) in HNEpCs. <b>B</b>. The percentage of total HNEpCs and J774.2 cells that are viable (Annexin V^-/7-AAD^-) is significantly reduced with LL-37 treatment, and this is dose-dependently reversed with GM-0111. <b>C</b>. The percentage of cells undergoing early death (Annexin V⁺/7-AAD^-) is significantly increased in HNEpCs treated with LL-37, and the effect is reversed with GM-0111. <b>D</b>. The percentage of HNEpCs and J774.2 in late cell death (Annexin V⁺/7-AAD⁺) is also significantly increased with LL-37 treatment and dose-dependently reversed with GM-0111. ****<i>P</i> ≤ 0.0001, ***<i>P</i> ≤ 0.001, **<i>P</i> ≤ 0.01, *<i>P</i> ≤ 0.05, ns (not significant) <i>P</i> > 0.05. The data represent the means ± SD (n = 4).</p

LL-37 induces morphologic change in HNEpCs (upper two rows) and J774.2 cells (bottom two rows), which are prevented with GM-0111 treatment.

<p>All images are at 40x magnification.</p