In situ photo-crosslinkable hyaluronic acid-based hydrogel embedded with GHK peptide nanofibers for bioactive wound healing.

A versatile hydrogel was developed for enhancing bioactive wound healing by introducing the amphiphilic GHK peptide (GHK-C16) into a photo-crosslinkable tyramine-modified hyaluronic acid (HA-Ty). GHK-C16 self-assembled into GHK nanofibers (GHK NF) in HA-Ty solution, which underwent in situ gelation after the wound area was filled with precursor solution. Blue light irradiation (460-490 nm), with riboflavin phosphate as a photoinitiator, was used to trigger crosslinking, which enhanced the stability of the highly degradable hyaluronic acid and enabled sustained release of the nanostructured GHK derivatives. The hydrogels provided a microenvironment that promoted the proliferation of dermal fibroblasts and the activation of cytokines, leading to reduced inflammation and increased collagen expression during wound healing. The complexation of Cu2+ into GHK nanofibers resulted in superior wound healing capabilities compared with non-lipidated GHK peptide with a comparable level of growth factor (EGF). Additionally, nanostructured Cu-GHK improved angiogenesis through vascular endothelial growth factor (VEGF) activation, which exerted a synergistic therapeutic effect. Furthermore, in vivo wound healing experiments revealed that the Cu-GHK NF/HA-Ty hydrogel accelerated wound healing through densely packed remodeled collagen in the dermis and promoting the growth of denser fibroblasts. HA-Ty hydrogels incorporating GHK NF also possessed improved mechanical properties and a faster wound healing rate, making them suitable for advanced bioactive wound healing applications. STATEMENT OF SIGNIFICANCE: By combining photo-crosslinkable tyramine-modified hyaluronic acid with self-assembled Cu-GHK-C16 peptide nanofibers (Cu-GHK NF), the Cu-GHK NF/HA-Ty hydrogel offers remarkable advantages over conventional non-structured Cu-GHK for wound healing. It enhances cell proliferation, migration, and collagen remodeling-critical factors in tissue regeneration. The incorporation of GHK nanofibers complexed with copper ions imparts potent anti-inflammatory effects, promoting cytokine activation and angiogenesis during wound healing. The Cu-GHK NF/hydrogel's unique properties, including in situ photo-crosslinking, ensure high customization and potency in tissue regeneration, providing a cost-effective alternative to growth factors. In vivo experiments further validate its efficacy, demonstrating significant wound closure, collagen remodeling, and increased fibroblast density. Overall, the Cu-GHK NF/HA-Ty hydrogel represents an advanced therapeutic option for wound healing applications.

Beta IV spectrin inhibits the metastatic growth of melanoma by suppressing VEGFR2-driven tumor angiogenesis.

BACKGROUND: Tumor-associated angiogenesis mediates the growth and metastasis of most solid cancers. Targeted therapies of the VEGF pathways can effectively block these processes but often fail to provide lasting benefits due to acquired resistance and complications. RESULTS: Recently, we discovered ßIV -spectrin as a powerful regulator of angiogenesis and potential new target. We previously reported that ßIV -spectrin is dynamically expressed in endothelial cells (EC) to induce VEGFR2 protein turnover during development. Here, we explored how ßIV -spectrin influences the tumor vasculature using the murine B16 melanoma model and determined that loss of EC-specific ßIV -spectrin dramatically promotes tumor growth and metastasis. Intraperitoneally injected B16 cells formed larger tumors with increased tumor vessel density and greater propensity for metastatic spread particularly to the chest cavity and lung compared to control mice. These results support ßIV -spectrin as a key regulator of tumor angiogenesis and a viable vascular target in cancer.

Endothelial tip/stalk cell selection requires BMP9-induced ßIV-spectrin expression during sprouting angiogenesis.

ßIV-Spectrin is a membrane cytoskeletal protein with specialized roles in the nervous system and heart. Recent evidence also indicates a fundamental role for ßIV-spectrin in angiogenesis as its endothelial-specific gene deletion in mice enhances embryonic lethality due to hypervascularization and hemorrhagic defects. During early vascular sprouting, ßIV-spectrin is believed to inhibit tip cell sprouting in favor of the stalk cell phenotype by mediating VEGFR2 internalization and degradation. Despite these essential roles, mechanisms governing ßIV-spectrin expression remain unknown. Here we identify bone morphogenetic protein 9 (BMP9) as a major inducer of ßIV-spectrin gene expression in the vascular system. We show that BMP9 signals through the ALK1/Smad1 pathway to induce ßIV-spectrin expression, which then recruits CaMKII to the cell membrane to induce phosphorylation-dependent VEGFR2 turnover. Although BMP9 signaling promotes stalk cell behavior through activation of hallmark stalk cell genes ID-1/3 and Hes-1 and Notch signaling cross-talk, we find that ßIV-spectrin acts upstream of these pathways as loss of ßIV-spectrin in neonate mice leads to retinal hypervascularization due to excessive VEGFR2 levels, increased tip cell populations, and strong Notch inhibition irrespective of BMP9 treatment. These findings demonstrate ßIV-spectrin as a BMP9 gene target critical for tip/stalk cell selection during nascent vessel sprouting.

ßIV-spectrin as a stalk cell-intrinsic regulator of VEGF signaling.

Defective angiogenesis underlies over 50 malignant, ischemic and inflammatory disorders yet long-term therapeutic applications inevitably fail, thus highlighting the need for greater understanding of the vast crosstalk and compensatory mechanisms. Based on proteomic profiling of angiogenic endothelial components, here we report ßIV-spectrin, a non-erythrocytic cytoskeletal protein, as a critical regulator of sprouting angiogenesis. Early loss of endothelial-specific ßIV-spectrin promotes embryonic lethality in mice due to hypervascularization and hemorrhagic defects whereas neonatal depletion yields higher vascular density and tip cell populations in developing retina. During sprouting, ßIV-spectrin expresses in stalk cells to inhibit their tip cell potential by enhancing VEGFR2 turnover in a manner independent of most cell-fate determining mechanisms. Rather, ßIV-spectrin recruits CaMKII to the plasma membrane to directly phosphorylate VEGFR2 at Ser984, a previously undefined phosphoregulatory site that strongly induces VEGFR2 internalization and degradation. These findings support a distinct spectrin-based mechanism of tip-stalk cell specification during vascular development.

Regulation of mitochondrial fission by GIPC-mediated Drp1 retrograde transport.

Dynamin-related protein 1 (Drp1) is a key regulator of mitochondrial fission, a large cytoplasmic GTPase recruited to the mitochondrial surface via transmembrane adaptors to initiate scission. While Brownian motion likely accounts for the local interactions between Drp1 and the mitochondrial adaptors, how this essential enzyme is targeted from more distal regions like the cell periphery remains unknown. Based on proteomic interactome screening and cell-based studies, we report that GAIP/RGS19-interacting protein (GIPC) mediates the actin-based retrograde transport of Drp1 toward the perinuclear mitochondria to enhance fission. Drp1 interacts with GIPC through its atypical C-terminal PDZ-binding motif. Loss of this interaction abrogates Drp1 retrograde transport resulting in cytoplasmic mislocalization and reduced fission despite retaining normal intrinsic GTPase activity. Functionally, we demonstrate that GIPC potentiates the Drp1-driven proliferative and migratory capacity in cancer cells. Together, these findings establish a direct molecular link between altered GIPC expression and Drp1 function in cancer progression and metabolic disorders.

Filamentous anti-influenza agents wrapping around viruses.

Owing to the emerging resistance to current anti-influenza therapies, strategies for blocking virus-cell interaction with agents that mimic interactions with host cell receptors are garnering interest. In this context, a multivalent presentation of sialyl groups on various types of scaffold materials such as dendrimers, liposomes, nanoparticles, and natural/synthetic polymers has been investigated for the inhibition of influenza A virus infection. However, the development of versatile antiviral agents based on monodisperse scaffolds capable of precise molecular design remains challenging. Whether an anisotropically extended filamentous nanostructure can serve as an effective scaffold for maximum inhibition of viral cell attachment has not been investigated. In this study, the preparation of a series of sialyllactose-conjugated filamentous bacteriophages (SLPhages), with controlled loading levels, ligand valencies, and two types of sialyllactose (α2,3' and α2,6'), is demonstrated. With optimal ligand loading and valency, SLPhages showed inhibitory activity (in vitro) against influenza A viruses at concentrations of tens of picomolar. This remarkable inhibition is due to the strong interaction between the SLPhage and the virus; this interaction is adequately potent to compensate for the cost of the bending and wrapping of the SLPhage around the influenza virus. Our study may open new avenues for the development of filamentous anti-viral agents, in which virus-wrapping or aggregation is the primary feature responsible for the blocking of cell entry.

Insulin Induces Microtubule Stabilization and Regulates the Microtubule Plus-end Tracking Protein Network in Adipocytes.

Insulin-stimulated glucose uptake is known to involve microtubules, although the function of microtubules and the microtubule-regulating proteins involved in insulin action are poorly understood. CLASP2, a plus-end tracking microtubule-associated protein (+TIP) that controls microtubule dynamics, was recently implicated as the first +TIP associated with insulin-regulated glucose uptake. Here, using protein-specific targeted quantitative phosphoproteomics within 3T3-L1 adipocytes, we discovered that insulin regulates phosphorylation of the CLASP2 network members G2L1, MARK2, CLIP2, AGAP3, and CKAP5 as well as EB1, revealing the existence of a previously unknown microtubule-associated protein system that responds to insulin. To further investigate, G2L1 interactome studies within 3T3-L1 adipocytes revealed that G2L1 coimmunoprecipitates CLASP2 and CLIP2 as well as the master integrators of +TIP assembly, the end binding (EB) proteins. Live-cell total internal reflection fluorescence microscopy in adipocytes revealed G2L1 and CLASP2 colocalize on microtubule plus-ends. We found that although insulin increases the number of CLASP2-containing plus-ends, insulin treatment simultaneously decreases CLASP2-containing plus-end velocity. In addition, we discovered that insulin stimulates redistribution of CLASP2 and G2L1 from exclusive plus-end tracking to "trailing" behind the growing tip of the microtubule. Insulin treatment increases α-tubulin Lysine 40 acetylation, a mechanism that was observed to be regulated by a counterbalance between GSK3 and mTOR, and led to microtubule stabilization. Our studies introduce insulin-stimulated microtubule stabilization and plus-end trailing of +TIPs as new modes of insulin action and reveal the likelihood that a network of microtubule-associated proteins synergize to coordinate insulin-regulated microtubule dynamics.

Synergetic roles of TGF-ß signaling in tissue engineering.

Recent advances in tissue engineering highlight biomaterial designs with context-specific growth factors, cytokines and various small molecules to better mimic the natural extracellular matrix (ECM) microenvironments. These efforts have led to direct improvements in cell-cell and cell-ECM interactions while mitigating undesirable cellular and immunogenic responses. In this short review, we focus on the crucial roles and regulation of transforming growth factor ß (TGF-ß) signaling in biomaterial applications during tissue repair and regeneration.

IR-783 Labeling of a Peptide Receptor for 'Turn-On' Fluorescence Based Sensing.

In this study, we examine a means for developing near-IR fluorescent sensors through streamlined, site-specific coupling with peptide-based receptors. As the penultimate step of solid-phase synthesis of a peptide-based receptor, we show a simple means of labeling the N' terminus with the near IR fluorophore IR-783 to afford a viable fluorescent sensor after cleavage from the resin. The proof-of-concept probe utilized a biotin mimetic peptide sequence as the receptive moiety. Here we revealed a "turn-on" fluorescence enhancement upon binding of the biotin mimetic probe to its intended streptavidin target. Not all peptide-receptive moieties tested were able to generate such an enhancement upon target binding, and as such, the rationale for the observed fluorescence response properties is discussed.

Microfabrication of Custom Collagen Structures Capable of Guiding Cell Morphology and Alignment.

The patterning of biological components into structural analogues of native tissues to simulate an environment for directing cell growth is one important strategy in biomaterials fabrication. It is widely accepted that chemical, mechanical, and topological cues from the extracellular matrix (ECM) provide important signals for guiding cells to exhibit characteristic polarity, orientation, and morphology. To fully understand the delicate relationship between cell behavior and ECM features, biomaterials fabrication requires improved techniques for tailoring nano/microstructured patterns from relevant biological building blocks rather than using nonbiological materials. Here we reveal a unique approach for the nano/microfabrication of custom patterned biomaterials using collagen as the sole building material. With this new fabrication technique, we further revealed that custom collagen patterns could direct the orientation and morphology of fibroblast growth as a function of vertex density and pattern spacing. Our findings suggest that this technique may be readily adopted for the free form fabrication of custom cell scaffolds purely from natural biological molecules including collagen, among other relevant ECM components.

Virus-based surface patterning of biological molecules, probes, and inorganic materials.

An essential requirement for continued technological advancement in many areas of biology, physics, chemistry, and materials science is the growing need to generate custom patterned materials. Building from recent achievements in the site-specific modification of virus for covalent surface tethering, we show in this work that stable 2D virus patterns can be generated in custom geometries over large area glass surfaces to yield templates of biological, biochemical, and inorganic materials in high density. As a nanomaterial building block, filamentous viruses have been extensively used in recent years to produce materials with interesting properties, owing to their ease of genetic and chemical modification. By utilizing un-natural amino acids generated at specific locations on the filamentous fd bacteriophage protein coat, surface immobilization is carried out on APTES patterned glass resulting in precise geometries of covalently linked virus material. This technique facilitated the surface display of a high density of virus that were labeled with biomolecules, fluorescent probes, and gold nanoparticles, thereby opening the possibility of integrating virus as functional components for surface engineering.

Responsive 3D microstructures from virus building blocks.

Fabrication of 3D biological structures reveals dynamic response to external stimuli. A liquid-crystalline bridge extrusion technique is used to generate 3D structures allowing the capture of Rayleigh-like instabilities, facilitating customization of smooth, helical, or undulating periodic surface textures. By integrating intrinsic biochemical functionality and synthetic components into controlled structures, this strategy offers a new form of adaptable materials.

Controlled surface immobilization of viruses via site-specific enzymatic modification.

A unique method for the controlled attachment of viruses (or other protein based materials) to a range of surfaces is revealed through site-specific linkages engineered at the fd phage p3 protein coat. After genetic encoding of the virus coat position that is to be engineered, enzymatic modification with a formylglycine generating enzyme (FGE) affords the conversion of a single amino acid at a precise location to yield a reactive aldehyde group. By implementing this modification at a specific p3 coat position, we demonstrate the ability to control the directed immobilization of the virus selectively onto amine exposed surfaces including APTES treated glass, polymeric supports, and protein coated magnetic beads. While the immobilized virus remains stable for even a month, we also show by controlled release from the surface that liberated viruses retain their infectivity. The adaptability of this modification strategy for virus engineering is demonstrated showing great potential for bioconjugation with a range of amine functionalized chemical targets. This is expected to greatly enhance the possibilities for future virus based materials and related technologies.