Immunohistochemical localization of fibronectin as a tool for the age determination of human skin wounds

We analyzed the distribution of fibronectin in routinely embedded tissue specimens from 53 skin wounds and 6 postmortem wounds. In postmortem wounds a faint but focal positive staining was exclusively found at the margin of the specimens which dit not extend into the adjacent stroma. Vital wounds were classified into 3 groups. The first comprising lesions with wound ages ranging from a few seconds to 30 min, the second comprising those with wound ages upt to 3 weeks, and the third group with lesions more than 3 weeks old. Ten out of 17 lesions with a wound age up to 30 min showed a clear positive reaction within the wound area. Three specimens in this group were completely negative, while in 4 additional cases the result was not significantly different from postmortem lesions. These 7 cases were characterized by acute death with extremely short survival times (only seconds). In wounds up to 3 weeks old fibronectin formed a distinct network containing an increasing number of inflammatory cells corresponding to the wound age. In 2 cases with a survival time of 17 days and in all wounds older than 3 weeks fibronectin was restricted to the surface of fibroblasts and to parallel arranged fibers in the granulation tissue without any network structures. We present evidence that fibronectin is a useful marker for vital wounds with a survival time of more than a few minutes. Fibronectin appears before neutrophilic granulocytes migrate into the wound area. Since a faint positive fibronectin staining is seen in postmortem lesions and bleedings, we propose that only those wounds which show strong positive fibronectin staining also extending into the adjacent stroma should be regarded as vital

Metastasis of Tumor Cells Is Enhanced by Downregulation of Bit1

Resistance to anoikis, which is defined as apoptosis induced by loss of integrin-mediated cell attachment to the extracellular matrix, is a determinant of tumor progression and metastasis. We have previously identified the mitochondrial Bit1 (Bcl-2 inhibitor of transcription) protein as a novel anoikis effector whose apoptotic function is independent from caspases and is uniquely controlled by integrins. In this report, we examined the possibility that Bit1 is suppressed during tumor progression and that Bit1 downregulation may play a role in tumor metastasis.Using a human breast tumor tissue array, we found that Bit1 expression is suppressed in a significant fraction of advanced stages of breast cancer. Targeted disruption of Bit1 via shRNA technology in lowly aggressive MCF7 cells conferred enhanced anoikis resistance, adhesive and migratory potential, which correlated with an increase in active Extracellular kinase regulated (Erk) levels and a decrease in Erk-directed phosphatase activity. These pro-metastasis phenotypes were also observed following downregulation of endogenous Bit1 in Hela and B16F1 cancer cell lines. The enhanced migratory and adhesive potential of Bit1 knockdown cells is in part dependent on their high level of Erk activation since down-regulating Erk in these cells attenuated their enhanced motility and adhesive properties. The Bit1 knockdown pools also showed a statistically highly significant increase in experimental lung metastasis, with no differences in tumor growth relative to control clones in vivo using a BALB/c nude mouse model system. Importantly, the pulmonary metastases of Bit1 knockdown cells exhibited increased phospho-Erk staining.These findings indicate that downregulation of Bit1 conferred cancer cells with enhanced anoikis resistance, adhesive and migratory properties in vitro and specifically potentiated tumor metastasis in vivo. These results underscore the therapeutic importance of restoring Bit1 expression in cancer cells to circumvent metastasis at least in part through inhibition of the Erk pathway

Modulation of functional pendant chains within poly(ethylene glycol) hydrogels for refined control of protein release

Hydrogels are highly attractive delivery vehicles for therapeutic proteins. Their innate biocompatibility, hydrophilicity and aqueous permeability allow stable encapsulation and release of proteins. The release rates also can be controlled simply by altering the crosslinking density of the polymeric network. However, the crosslinking density also influences the mechanical properties of hydrogels, generally opposite to the permeability. In addition, the release of larger proteins may be hindered below critically diminished porosity determined by the crosslinking density. Herein, the physical properties of the hydrogels are tuned by presenting functional pendant chains, independent of crosslinking density. Heterobifunctional poly(ethylene glycol) monomethacrylate (PEGMA) with various end functional groups is synthesized and copolymerized with PEG dimethacrylate (PEGDA) to engineer PEG hydrogels with pendant PEG chains. The pendant chains of the PEG hydrogels consisting of sulfonate, trimethylammonium chloride, and phenyl groups are utilized to provide negative charge, positive charge and hydrophobicity, respectively, to the hydrogels. The release rates of proteins with different isoelectric points are controlled in a wide range by the type and the density of functional pendant chains via electrostatic and hydrophobic interactions

Self-Assembling Peptide Nanofiber Scaffolds Accelerate Wound Healing

Cutaneous wound repair regenerates skin integrity, but a chronic failure to heal results in compromised tissue function and increased morbidity. To address this, we have used an integrated approach, using nanobiotechnology to augment the rate of wound reepithelialization by combining self-assembling peptide (SAP) nanofiber scaffold and Epidermal Growth Factor (EGF). This SAP bioscaffold was tested in a bioengineered Human Skin Equivalent (HSE) tissue model that enabled wound reepithelialization to be monitored in a tissue that recapitulates molecular and cellular mechanisms of repair known to occur in human skin. We found that SAP underwent molecular self-assembly to form unique 3D structures that stably covered the surface of the wound, suggesting that this scaffold may serve as a viable wound dressing. We measured the rates of release of EGF from the SAP scaffold and determined that EGF was only released when the scaffold was in direct contact with the HSE. By measuring the length of the epithelial tongue during wound reepithelialization, we found that SAP scaffolds containing EGF accelerated the rate of wound coverage by 5 fold when compared to controls without scaffolds and by 3.5 fold when compared to the scaffold without EGF. In conclusion, our experiments demonstrated that biomaterials composed of a biofunctionalized peptidic scaffold have many properties that are well-suited for the treatment of cutaneous wounds including wound coverage, functionalization with bioactive molecules, localized growth factor release and activation of wound repair

Normalization of tumour blood vessels improves the delivery of nanomedicines in a size-dependent manner

The blood vessels of cancerous tumours are leaky and poorly organized. This can increase the interstitial fluid pressure inside tumours and reduce blood supply to them, which impairs drug delivery. Anti-angiogenic therapies—which ‘normalize’ the abnormal blood vessels in tumours by making them less leaky—have been shown to improve the delivery and effectiveness of chemotherapeutics with low molecular weights, but it remains unclear whether normalizing tumour vessels can improve the delivery of nanomedicines. Here, we show that repairing the abnormal vessels in mammary tumours, by blocking vascular endothelial growth factor receptor-2, improves the delivery of smaller nanoparticles (diameter, 12 nm) while hindering the delivery of larger nanoparticles (diameter, 125 nm). Using a mathematical model, we show that reducing the sizes of pores in the walls of vessels through normalization decreases the interstitial fluid pressure in tumours, thus allowing small nanoparticles to enter them more rapidly. However, increased steric and hydrodynamic hindrances, also associated with smaller pores, make it more difficult for large nanoparticles to enter tumours. Our results further suggest that smaller (~12 nm) nanomedicines are ideal for cancer therapy due to their superior tumour penetration.ImClone Systems IncorporatedNational Institutes of Health (U.S.) (P01-CA080124)National Institutes of Health (U.S.) (R01-CA126642)National Institutes of Health (U.S.) (R01-CA115767)National Institutes of Health (U.S.) (R01-CA096915)National Institutes of Health (U.S.) (R01-CA085140)National Institutes of Health (U.S.) (R01-CA098706)National Institutes of Health (U.S.) (T32-CA073479)United States. Dept. of Defense (Breast Cancer Research Innovator Award W81XWH-10-1-0016

Cytotoxic targeting of F9 teratocarcinoma tumours with anti-ED-B fibronectin scFv antibody modified liposomes

We prepared small unilamellar liposomes derivatised with single chain antibody fragments specific for the ED-B domain of B-fibronectin. This extracellular matrix associated protein is expressed around newly forming blood vessels in the vicinity of many types of tumours. The single chain antibody fragments were functionalised by introduction of C-terminal cysteines and linked to liposomes via maleimide groups located at the terminal ends of poly(ethylene glycol) modified phospholipids. The properties of these anti-ED-B single chain antibody fragments-liposomes were analysed in vitro on ED-B fibronectin expressing Caco-2 cells and in vivo by studying their biodistribution and their therapeutic potential in mice bearing subcutanous F9 teratocarcinoma tumours. Radioactively labelled (114mIndium) single chain antibody fragments-liposomes accumulated in the tumours at 2–3-fold higher concentrations during the first 2 h after i.v. injection compared to unmodified liposomes. After 6–24 h both liposome types were found in similar amounts (8–10% injected dose g−1) in the tumours. Animals treated i.v. with single chain antibody fragments-liposomes containing the new cytotoxic agent 2′-deoxy-5-fluorouridylyl-N4-octadecyl-1-β-D-arabinofuranosylcytosine (30 mg kg-1 per dose, five times every 24 h) showed a reduction of tumour growth by 62–90% determined on days 5 and 8, respectively, compared to animals receiving control liposomes. Histological analysis revealed a marked reduction of F9 tumour cells and excessive deposition of fibronectin in the extracellular matrix after treatment with single chain antibody fragments-2-dioxy-5-fluorouridylyl-N4-octadecyl-1-β-D-arabinofuranosylcytosine-liposomes. Single chain antibody fragments-liposomes targeted to ED-B fibronectin positive tumours therefore represent a promising and versatile novel drug delivery system for the treatment of tumours

Sustained proliferation in cancer: mechanisms and novel therapeutic targets

Proliferation is an important part of cancer development and progression. This is manifest by altered expression and/or activity of cell cycle related proteins. Constitutive activation of many signal transduction pathways also stimulates cell growth. Early steps in tumor development are associated with a fibrogenic response and the development of a hypoxic environment which favors the survival and proliferation of cancer stem cells. Part of the survival strategy of cancer stem cells may manifested by alterations in cell metabolism. Once tumors appear, growth and metastasis may be supported by overproduction of appropriate hormones (in hormonally dependent cancers), by promoting angiogenesis, by undergoing epithelial to mesenchymal transition, by triggering autophagy, and by taking cues from surrounding stromal cells. A number of natural compounds (e.g., curcumin, resveratrol, indole-3-carbinol, brassinin, sulforaphane, epigallocatechin-3-gallate, genistein, ellagitannins, lycopene and quercetin) have been found to inhibit one or more pathways that contribute to proliferation (e.g., hypoxia inducible factor 1, nuclear factor kappa B, phosphoinositide 3 kinase/Akt, insulin-like growth factor receptor 1, Wnt, cell cycle associated proteins, as well as androgen and estrogen receptor signaling). These data, in combination with bioinformatics analyses, will be very important for identifying signaling pathways and molecular targets that may provide early diagnostic markers and/or critical targets for the development of new drugs or drug combinations that block tumor formation and progression