Pharmacologic inhibition of RGD-binding integrins ameliorates fibrosis and improves function following kidney injury

Fibrosis is a final common pathway for many causes of progressive chronic kidney disease (CKD). Arginine-glycine-aspartic acid (RGD)-binding integrins are important mediators of the pro-fibrotic response by activating latent TGF-β at sites of injury and by providing myofibroblasts information about the composition and stiffness of the extracellular matrix. Therefore, blockade of RGD-binding integrins may have therapeutic potential for CKD. To test this idea, we used small-molecule peptidomimetics that potently inhibit a subset of RGD-binding integrins in a murine model of kidney fibrosis. Acute kidney injury leading to fibrosis was induced by administration of aristolochic acid. Continuous subcutaneous administration of CWHM-12, an RGD integrin antagonist, for 28 days improved kidney function as measured by serum creatinine. CWHM-12 significantly reduced Collagen 1 (Col1a1) mRNA expression and scar collagen deposition in the kidney. Protein and gene expression markers of activated myofibroblasts, a major source of extracellular matrix deposition in kidney fibrosis, were diminished by treatment. RNA sequencing revealed that inhibition of RGD integrins influenced multiple pathways that determine the outcome of the response to injury and of repair processes. A second RGD integrin antagonist, CWHM-680, administered once daily by oral gavage was also effective in ameliorating fibrosis. We conclude that targeting RGD integrins with such small-molecule antagonists is a promising therapeutic approach in fibrotic kidney disease

Cell response to RGD density in cross-linked artificial extracellular matrix protein films

This study examines the adhesion, spreading, and migration of human umbilical vein endothelial cells on cross-linked films of artificial extracellular matrix (aECM) proteins. The aECM proteins described here were designed for application in small-diameter grafts and are composed of elastin-like structural repeats and fibronectin cell-binding domains. aECM-RGD contains the RGD sequence derived from fibronectin; the negative control protein aECM-RDG contains a scrambled cell-binding domain. The covalent attachment of poly(ethylene glycol) (PEG) to aECM substrates reduced nonspecific cell adhesion to aECM-RDG-PEG but did not preclude sequence-specific adhesion of endothelial cells to aECM-RGD-PEG. Variation in ligand density was accomplished by the mixing of aECM-RGD-PEG and aECM-RDG-PEG prior to cross-linking. Increasing the density of RGD domains in cross-linked films resulted in more robust cell adhesion and spreading but did not affect cell migration speed. Control of cell-binding domain density in aECM proteins can thus be used to modulate cell adhesion and spreading and will serve as an important design tool as these materials are further developed for use in surgery, tissue engineering, and regenerative medicine

YB-1 dependent oncolytic adenovirus efficiently inhibits tumor growth of glioma cancer stem like cells

Background: The brain cancer stem cell (CSC) model describes a small subset of glioma cells as being responsible for tumor initiation, conferring therapy resistance and tumor recurrence. In brain CSC, the PI3-K/AKT and the RAS/mitogen activated protein kinase (MAPK) pathways are found to be activated. In consequence, the human transcription factor YB-1, knowing to be responsible for the emergence of drug resistance and driving adenoviral replication, is phosphorylated and activated. With this knowledge, YB-1 was established in the past as a biomarker for disease progression and prognosis. This study determines the expression of YB-1 in glioblastoma (GBM) specimen in vivo and in brain CSC lines. In addition, the capacity of Ad-Delo3-RGD, an YB-1 dependent oncolytic adenovirus, to eradicate CSC was evaluated both in vitro and in vivo. Methods: YB-1 expression was investigated by immunoblot and immuno-histochemistry. In vitro, viral replication as well as the capacity of Ad-Delo3-RGD to replicate in and, in consequence, to kill CSC was determined by real-time PCR and clonogenic dilution assays. In vivo, Ad-Delo3-RGD-mediated tumor growth inhibition was evaluated in an orthotopic mouse GBM model. Safety and specificity of Ad-Delo3-RGD were investigated in immortalized human astrocytes and by siRNA-mediated downregulation of YB-1. Results: YB-1 is highly expressed in brain CSC lines and in GBM specimen. Efficient viral replication in and virus-mediated lysis of CSC was observed in vitro. Experiments addressing safety aspects of Ad-Delo3-RGD showed that (i) virus production in human astrocytes was significantly reduced compared to wild type adenovirus (Ad-WT) and (ii) knockdown of YB-1 significantly reduced virus replication. Mice harboring othotopic GBM developed from a temozolomide (TMZ)-resistant GBM derived CSC line which was intratumorally injected with Ad-Delo3-RGD survived significantly longer than mice receiving PBS-injections or TMZ treatment. Conclusion: The results of this study supported YB-1 based virotherapy as an attractive therapeutic strategy for GBM treatment which will be exploited further in multimodal treatment concepts

Synthesis and Cell Adhesive Properties of Linear and Cyclic RGD Functionalized Polynorbornene Thin Films

Described herein is the efficient synthesis and evaluation of bioactive arginine-glycine-aspartic acid (RGD) functionalized polynorbornene-based materials for cell adhesion and spreading. Polynorbornenes containing either linear or cyclic RGD peptides were synthesized by ring-opening metathesis polymerization (ROMP) using the well-defined ruthenium initiator [(H_(2)IMes)(pyr)_(2)(Cl)_(2)Ru═CHPh]. The random copolymerization of three separate norbornene monomers allowed for the incorporation of water-soluble polyethylene glycol (PEG) moieties, RGD cell recognition motifs, and primary amines for postpolymerization cross-linking. Following polymer synthesis, thin-film hydrogels were formed by cross-linking with bis(sulfosuccinimidyl) suberate (BS^3), and the ability of these materials to support human umbilical vein endothelial cell (HUVEC) adhesion and spreading was evaluated and quantified. When compared to control polymers containing either no peptide or a scrambled RDG peptide, polymers with linear or cyclic RGD at varying concentrations displayed excellent cell adhesive properties in both serum-supplemented and serum-free media. Polymers with cyclic RGD side chains maintained cell adhesion and exhibited comparable integrin binding at a 100-fold lower concentration than those carrying linear RGD peptides. The precise control of monomer incorporation enabled by ROMP allows for quantification of the impact of RGD structure and concentration on cell adhesion and spreading. The results presented here will serve to guide future efforts for the design of RGD functionalized materials with applications in surgery, tissue engineering, and regenerative medicine

Evidence that an RGD-dependent receptor mediates the binding of oligodendrocytes to a novel ligand in a glial-derived matrix.

A simple adhesion assay was used to measure the interaction between rat oligodendrocytes and various substrata, including a matrix secreted by glial cells. Oligodendrocytes bound to surfaces coated with fibronectin, vitronectin and a protein component of the glial matrix. The binding of cells to all of these substrates was inhibited by a synthetic peptide (GRGDSP) modeled after the cell-binding domain of fibronectin. The component of the glial matrix responsible for the oligodendrocyte interaction is a protein which is either secreted by the glial cells or removed from serum by products of these cultures; serum alone does not promote adhesion to the same extent as the glial-derived matrix. The interaction of cells with this glial-derived matrix requires divalent cations and is not mediated by several known RGD-containing extracellular proteins, including fibronectin, vitronectin, thrombospondin, type I and type IV collagen, and tenascin

Integrin-mediated membrane blebbing is dependent on the NHE1 and NCX1 activities.

Integrin-mediated signal transduction and membrane blebbing have been well studied to modulate cell adhesion, spreading and migration^1-6^. However, the relationship between membrane blebbing and integrin signaling has not been explored. Here we show that integrin-ligand interaction induces membrane blebbing and membrane permeability change. We found that sodium-proton exchanger 1 (NHE1) and sodium-calcium exchanger 1 (NCX1) are located in the membrane blebbing sites and inhibition of NHE1 disrupts membrane blebbing and decreases membrane permeability change. However, inhibition of NCX1 enhances cell blebbing to cause cell swelling which is correlated with an intracellular sodium accumulation induced by NHE17. These data suggest that sodium influx induced by NHE1 is a driving force for membrane blebbing growth, while sodium efflux induced by NCX1 in a reverse mode causes membrane blebbing retraction. Together, these data reveal a novel function of NHE1 and NCX1 in membrane permeability change and blebbing and provide the link for integrin signaling and membrane blebbing

A point mutation of integrin beta 1 subunit blocks binding of alpha 5 beta 1 to fibronectin and invasin but not recruitment to adhesion plaques.

A point mutation in a highly conserved region of the beta 1 subunit, Asp130 to Ala (D130A) substitution, abrogates the Arg-Gly-Asp (RGD)-dependent binding of alpha 5 beta 1 to fibronectin (FN) without disrupting gross structure or heterodimer assembly. The D130A mutation also interferes with binding to invasin, a ligand that lacks RGD sequence. In spite of the lack of detectable FN binding by alpha 5 beta 1(D130A), it was recruited to adhesion plaques formed on FN by endogenous hamster receptors. Thus, intact ligand binding function is not required for recruitment of alpha 5 beta 1 to adhesion plaques. Overexpression of beta 1(D130A) partially interfered with endogenous alpha 5 beta 1 function, thus defining a dominant negative beta 1 integrin mutation

Use of the R-group descriptor for alignment-free QSAR

An R-group descriptor characterises the distribution of some atom-based property, such as elemental type or partial atomic charge, at increasing numbers of bonds distant from the point of substitution on a parent ring system. Application of Partial Least Squares (PLS) to datasets for which bioactivity data and R-group descriptor information are available is shown to provide an effective way of generating QSAR models with a high level of predictive ability. The resulting models are competitive with the models produced by established QSAR approaches, are readily interpretable in structural terms, and are shown to be of value in the optimisation of a lead series