Although, drugs are required in the various skin compartments such as viable epidermis, dermis, or hair follicles, to efficiently treat skin diseases, drug delivery into and across the skin is still challenging. An improved understanding of skin barrier physiology is mandatory to optimize drug penetration and permeation. The various barriers of the skin have to be known in detail, which means methods are needed to measure their functionality and outside-in or inside-out passage of molecules through the various barriers. In this review, we summarize our current knowledge about mechanical barriers, i.e., stratum corneum and tight junctions, in interfollicular epidermis, hair follicles and glands. Furthermore, we discuss the barrier properties of the basement membrane and dermal blood vessels. Barrier alterations found in skin of patients with atopic dermatitis are described. Finally, we critically compare the up-to-date applicability of several physical, biochemical and microscopic methods such as transepidermal water loss, impedance spectroscopy, Raman spectroscopy, immunohistochemical stainings, optical coherence microscopy and multiphoton microscopy to distinctly address the different barriers and to measure permeation through these barriers in vitro and in vivo.

Objective—: Inflammatory conditions provoke essential processes in the human vascular system. It leads to the formation of ultralarge von Willebrand factor (VWF) fibers, which are immobilized on the endothelial cell surface and transform to highly adhesive strings under shear conditions. Furthermore, leukocytes release a meshwork of DNA (neutrophil extracellular traps) during the process of the recently discovered cell death program NETosis. In the present study, we characterized the interaction between VWF and DNA and possible binding sites to underline the role of VWF in thrombosis and inflammation besides its function in platelet adhesion. Approach and Results—: Both functionalized surfaces and intact cell layers of human umbilical vein endothelial cells were perfused with isolated, protein-free DNA or leukocytes from whole blood at distinct shear rates. DNA–VWF interaction was monitored using fluorescence microscopy, ELISA-based assays, molecular dynamics simulations, and electrostatic potential calculations. Isolated DNA, as well as DNA released by stimulated leukocytes, was able to bind to shear-activated, but not inactivated, VWF. However, DNA–VWF binding does not alter VWF degradation by a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13. Moreover, DNA–VWF interaction can be blocked using unfractionated and low-molecular-weight heparin, and DNA–VWF complexes attenuate platelet binding to VWF. These findings were supported using molecular dynamics simulations and electrostatic calculations of the A1- and A2-domains. Conclusions—: Our findings suggest that VWF directly binds and immobilizes extracellular DNA released from leukocytes. Therefore, we hypothesize that VWF might act as a linker for leukocyte adhesion to endothelial cells, supporting leukocyte extravasation and inflammation.

Tumor cells interact with blood constituents and these interactions promote metastasis. Selectins are vascular receptors facilitating interactions of tumor cells with platelets, leukocytes, and endothelium, but the role of endothelial E-selectin remains unclear. Here we show that E-selectin is a major receptor for monocyte recruitment to tumor cell–activated endothelium. Experimental and spontaneous lung metastasis using murine tumor cells, without E-selectin ligands, were attenuated in E-selectin–deficient mice. Tumor cell–derived CCL2 promoted endothelial activation, resulting in enhanced endothelial E-selectin expression. The recruitment of inflammatory monocytes to metastasizing tumor cells was dependent on the local endothelial activation and the presence of E-selectin. Monocytes promoted transendothelial migration of tumor cells through the induction of E-selectin–dependent endothelial retractions and a subsequent modulation of tight junctions through dephosphorylation of VE-cadherin. Thus, endothelial E-selectin shapes the tumor microenvironment through the recruitment, adhesion, and activation of monocytes that facilitate tumor cell extravasation and thereby metastasis. These findings provide evidence that endothelial E-selectin is a novel factor contributing to endothelial retraction required for efficient lung metastasis. Cancer Res; 76(18); 5302–12. ©2016 AACR.

Summary: The specific interactions of von Willebrand factor (VWF) with the vessel wall, platelets or other interfaces strongly depend on (a shearinduced) VWF activation. Shear flow has been shown to induce a conformational transition of VWF, but is modulated by its thermodynamic state (state-function relationship). The state in turn is determined by physical (e.g. vessel geometry), physico-chemical (e.g. pH) and molecular-biological (e.g. mutants, binding) factors. Combining established results with recent insights, we reconstruct VWF biology and its statefunction relationship from endothelial cell release to final degradation in the human vasculature. After VWF secretion, endothelial-anchored and shear activated VWF multimers can rapidly interact with surrounding colloids, typically with platelets. Simultaneously, this VWF activation enables ADAMTS13 to cleave VWF multimers thereby limiting VWF binding capacity. The subsequent cell-surface dissociation leads to a VWF recoiling to a globular conformation, shielding from further degradation by ADAMTS13. High local concentrations of these soluble VWF multimers, transported to the downstream vasculature, are capable for an immediate reactivation and re-polymerisation initiating colloid-binding or VWF-colloid aggregation at the site of inflamed endothelium, vessel injuries or pathological high-shear areas. Focusing on these functional steps in the lifecycle of VWF, its qualitative and quantitative deficiencies in the different VWD types will facilitate more precise diagnostics and reliable risk stratification for prophylactic therapies. The underlying biophysical principles are of general character, which broadens prospective studies on the physiological and pathophysiological impact of VWF and VWF-associated diseases and beares hope for a more universal understanding of an entire class of phenomena.

Blood clotting is a process by which a haemostatic plug is assembled at the site of injury. The formation of such a plug, which is essentially a (bio)polymer–colloid composite, is believed to be driven by shear flow in its initial phase, and contrary to our intuition, its assembly is enhanced under stronger flowing conditions. Here, inspired by blood clotting, we show that polymer–colloid composite assembly in shear flow is a universal process that can be tailored to obtain different types of aggregates including loose and dense aggregates, as well as hydrodynamically induced ‘log’-type aggregates. The process is highly controllable and reversible, depending mostly on the shear rate and the strength of the polymer–colloidbinding potential. Our results have important implications for the assembly of polymer–colloid composites, an important challenge of immense technological relevance. Furthermore, flow-driven reversible composite formation represents a new paradigm in non-equilibrium self-assembly.

The application of multiphoton microscopy in the field of biomedical research and advanced diagnostics promises unique insights into the pathophysiology of inflammatory skin diseases. In the present study, we combined multiphoton-based intravital tomography (MPT) and fluorescence lifetime imaging (MPT-FLIM) within the scope of a clinical trial of atopic dermatitis with the aim of providing personalised data on the aetiopathology of inflammation in a non-invasive manner at patients’ bedsides. These ‘optical biopsies’ generated via MPT were morphologically analysed and aligned with classical skin histology. Because of its subcellular resolution, MPT provided evidence of a redistribution of mitochondria in keratinocytes, indicating an altered cellular metabolism. Two independent morphometric algorithms reliably showed an even distribution in healthy skin and a perinuclear accumulation in inflamed skin. Moreover, using MPT-FLIM, detection of the onset and progression of inflammatory processes could be achieved. In conclusion, the change in the distribution of mitochondria upon inflammation and the verification of an altered cellular metabolism facilitate a better understanding of inflammatory skin diseases and may permit early diagnosis and therapy.