Human Stimulus Factor Is a Promising Peptide for Delivery of Therapeutics.

Fluticasone propionate uptake in the presence of a proprietary cell-penetrating peptide (human stimulus factor, [HSF]) based on the N-terminal domain of lactoferrin was studied, alone and in combination with salmeterol, using an air interface Calu-3 epithelial model. The HSF enhanced uptake and transport of fluticasone propionate across the epithelial barrier when alone and in presence of salmeterol. This was attributed to transcellular-mediated uptake. This HSF is a promising peptide for delivery of therapeutics where enhanced epithelial penetrating is required.

Inhaled gene delivery: a formulation and delivery approach.

INTRODUCTION: Gene therapy is a potential alternative to treat a number of diseases. Different hurdles are associated with aerosol gene delivery due to the susceptibility of plasmid DNA (pDNA) structure to be degraded during the aerosolization process. Different strategies have been investigated in order to protect and efficiently deliver pDNA to the lungs using non-viral vectors. To date, no successful therapy involving non-viral vectors has been marketed, highlighting the need for further investigation in this field. Areas covered: This review is focused on the formulation and delivery of DNA to the lungs, using non-viral vectors. Aerosol gene formulations are divided according to the current delivery systems for the lung: nebulizers, dry powder inhalers and pressurized metered dose inhalers; highlighting its benefits, challenges and potential application. Expert opinion: Successful aerosol delivery is achieved when the supercoiled DNA structure is protected during aerosolization. A formulation strategy or compounds that can protect, stabilize and efficiently transfect DNA into the cells is desired in order to produce an effective, low-cost and safe formulation. Nebulizers and dry powder inhalers are the most promising approaches to be used for aerosol delivery, due to the lower shear forces involved. In this context it is also important to highlight the importance of considering the 'pDNA-formulation-device system' as an integral part of the formulation development for a successful nucleic acid delivery.

Laminin-5-deficient human keratinocytes: defective adhesion results in a saltatory and inefficient mode of migration.

Laminin-5 is a major adhesion protein of the skin basement membrane and crucially involved in integrin-mediated cell substrate attachment of keratinocytes, which is important for hemidesmosomal anchorage as well as for keratinocyte migration during epidermal wound healing. To investigate its role in keratinocyte migration, we analyzed laminin-5-deficient cells of patients with a lethal variant of junctional epidermolysis bullosa. Normal migrating keratinocytes adopted monopolar morphology with a distinct front lamella and employed a continuous mode of translocation. In contrast, laminin-5-deficient cells assumed a stretched bipolar shape with two lamella regions and migrated in a discontinuous, saltatory manner characterized by significantly decreased directional persistence and reduced migration velocity. The distinct morphology as well as the migratory phenotype apparently resulted from a defect in the formation of cell substrate adhesions that were completely missing in the cell body and less stable in the lamella regions. Accordingly in normal keratinocytes, a bipolar shape and a saltatory migration mode were inducible by blocking laminin-5-mediated substrate adhesion. Our findings clearly point to an essential role of laminin-5 in forming dynamic cell substrate adhesion during migration of epidermal keratinocytes and provide an explanation for the cellular mechanisms that underlie the lethal form of junctional epidermolysis bullosa.

Impaired epidermal wound healing in vivo upon inhibition or deletion of Rac1.

To address the functions of Rac1 in keratinocytes of the basal epidermal layer and in the outer root sheath of hair follicles, we generated transgenic mice expressing a dominant inhibitory mutant of Rac, N17Rac1, under the control of the keratin 14 promoter. These mice do not exhibit an overt skin phenotype but show protracted skin wound re-epithelialization. Investigation into the underlying mechanisms revealed that in vivo both proliferation of wound-edge keratinocytes and centripetal migration of the neo-epidermis were impaired. Similar results were obtained in mice with an epidermis-specific deletion of Rac1. Primary epidermal keratinocytes that expressed the N17Rac1 transgene were less proliferative than control cells and showed reduced ERK1/2 phosphorylation upon growth factor stimulation. Adhesion, spreading, random migration and closure of scratch wounds in vitro were significantly inhibited on collagen I and, to a lesser extent, on fibronectin. Stroboscopic analysis of cell dynamics (SACED) of N17Rac1 transgenic and control keratinocytes identified decreased lamella-protrusion persistence in connection with increased ruffle frequency as a probable mechanism for the observed impairment of keratinocyte adhesion and migration. We conclude that Rac1 is functionally required for normal epidermal wound healing and, in this context, exerts a dual function - namely the regulation of keratinocyte proliferation and migration.

HaCaT keratinocytes secrete lysosomal cysteine proteinases during migration.

Cathepsin B, a lysosomal cysteine proteinase, was detected within vesicles of cellular protrusions forming cell-cell contact sites between keratinocytes of the stratum spinosum of human skin. This observation suggested the possibility that secretion of the protease into the pericellular spaces could be involved in the dissociation of cell-cell contacts to enable intraepidermal keratinocyte migration. To determine whether cathepsin B is indeed secreted from migrating keratinocytes, we first used subconfluent HaCaT cells as a culture model to study spontaneous keratinocyte migration. A cathepsin B-specific fluorescent affinity label proved the association of mature cathepsin B with the surfaces of HaCaT cells at the leading edges of growing cells. Second, we used scratch-wounds of confluent HaCaT monolayers as a model of induced keratinocyte migration. Cathepsin B was detected within lysosomes, i.e. vesicles within the perinuclear region of non-wounded cells. Expression of cathepsin B was up-regulated and cathepsin B-positive vesicles showed a redistribution from perinuclear to peripheral regions of keratinocytes at the wound margins within 4 h after wounding. Enzyme cytochemistry further showed that cell surface-associated cathepsin B was proteolytically active at the leading fronts of migrating keratinocytes. In addition, increased amounts of mature forms of cathepsin B were detected within the conditioned media of HaCaT cells during the first 4 h after scratch-wounding. In contrast, and as a control, the activity of the cytosolic enzyme lactate dehydrogenase was not significantly higher in media of wounded cells as compared with non-wounded controls, arguing for a specific induction of cathepsin B secretion upon wounding and migration of the cells. This was further substantiated by applying various cathepsin B-specific inhibitors after wounding. These experiments showed that the migration ability of keratinocytes was reduced due to the blockage of functional cathepsin B. Thus, our results strongly suggest that cell surface-associated cathepsin B is a protease that contributes to the remodelling of the extracellular matrix and thereby promotes keratinocyte migration during wound healing.