The endothelial glycocalyx and its regulated shedding are important to vascular health. Endo-β-D-glucuronidase heparanase-1 (HPSE1) is the only enzyme that can shed heparan sulfate. However, the mechanisms are not well understood. We show that HPSE1 activity aggravated Toll-like receptor 4 (TLR4)-mediated response of endothelial cells to LPS. On the contrary, overexpression of its endogenous inhibitor, heparanase-2 (HPSE2) was protective. The microfluidic chip flow model confirmed that HPSE2 prevented heparan sulfate shedding by HPSE1. Furthermore, heparan sulfate did not interfere with cluster of differentiation-14 (CD14)-dependent LPS binding, but instead reduced the presentation of the LPS to TLR4. HPSE2 reduced LPS-mediated TLR4 activation, subsequent cell signalling, and cytokine expression. HPSE2-overexpressing endothelial cells remained protected against LPS-mediated loss of cell-cell contacts. In vivo, expression of HPSE2 in plasma and kidney medullary capillaries was decreased in mouse sepsis model. We next applied purified HPSE2 in mice and observed decreases in TNFα and IL-6 plasma concentrations after intravenous LPS injections. Our data demonstrate the important role of heparan sulfate and the glycocalyx in endothelial cell activation and suggest a protective role of HPSE2 in microvascular inflammation. HPSE2 offers new options for protection against HPSE1-mediated endothelial damage and preventing microvascular disease.

High-resolution, high-speed 3D printing by two-photon polymerization (2PP) with a Nd:YVO 4 Q-switched microchip laser at its fundamental wavelength of 1064 nm is demonstrated. Polymerization scan speeds of up to 20 mm/s and feature sizes of 250 nm are achieved using a high repetition rate Q-switched microchip laser with a semiconductor saturable absorber mirror (SESAM) and photoresist with a new photo-initiator bearing 6dialkylaminobenzufuran as electron donor and indene-1,3-dione moiety as electron acceptor. The obtained results demonstrate the high potential of Q-switched microchip lasers for applications in 2PP 3D printing.

A technique to fabricate electrically conductive all-polymer 3D microstructures is reported. Superior conductivity, high spatial resolution and three-dimensionality are achieved by successive application of two-photon polymerization and in situ oxidative polymerization to a bi-component formulation, containing a photosensitive host matrix and an intrinsically conductive polymer precursor. By using polyethylene glycol diacrylate (PEG-DA) and 3,4-ethylenedioxythiophene (EDOT), the conductivity of 0.04 S/cm is reached, which is the highest value for the two-photon polymerized all-polymer microstructures to date. The measured electrical conductivity dependency on the EDOT concentration indicates percolation phenomenon and a three-dimensional nature of the conductive pathways. Tunable conductivity, biocompatibility, and environmental stability are the characteristics offered by PEG-DA/EDOT blends which can be employed in biomedicine, MEMS, microfluidics, and sensorics.

In this paper, approaches for the realization of high-resolution periodic structures with gap sizes at sub-100 nm scale by two-photon polymerization (2PP) are presented. The impact of laser intensity on the feature sizes and surface quality is investigated. The influence of different photosensitive materials on the structure formation is compared. Based on the elliptical geometry character of the voxel, the authors present an idea to realize high-resolution structures with feature sizes less than 100 nm by controlling the laser focus position with respect to the glass substrate. This investigation covers structures fabricated respectively in the plane along and perpendicular to the major axis of voxel. The authors also provide a useful approach to manage the fabrication of proposed periodic structure with a periodic distance of 200 nm and a gap size of 65 nm.

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Word count: 6827 Total number of figures and tables: 7 TOC category: Basic TOC subcategory: Arteriosclerosis, Thrombosis, and Vascular Biology 2 1 2 3 4 5 6 AbstractObjective. The endothelial glycocalyx and the regulation of its shedding are important to vascular health. Endo-β-D-glucuronidase heparanase-1 (HPSE1) is the only enzyme that can shed heparan sulfate. However, the mechanisms are not well understood.Approach and results. To investigate HPSE1 and its endogenous inhibitor, heparanase-2 (HPSE2), we used cell culture, lentiviral protein overexpression, a microfluidic chip model of cell culture under shear stress conditions, and lipopolysaccharide (LPS) injections in mice.We show that HPSE1 activity aggravated Toll-like receptor 4 (TLR4)-mediated response of endothelial cells to LPS. On the contrary, HPSE2 overexpression was protective. The microfluidic chip flow model confirmed that HPSE2 prevented heparan sulfate shedding by HPSE1. Furthermore, heparan sulfate did not interfere with cluster of differentiation-14 (CD14)-dependent LPS binding, but instead reduced the presentation of the LPS-CD14 complex to TLR4. HPSE2 reduced LPS-mediated TLR4 activation by LPS, subsequent cell signaling, and cytokine expression. Moreover, HPSE2-overexpressing endothelial cells remained protected against LPS-mediated loss of cell-cell contacts. In vivo, expression of HPSE2 in plasma and kidney medullary capillaries was decreased in mouse sepsis model. We next applied purified HPSE2 in mice and observed decreases in TNFα and IL-6 plasma concentrations after intravenous LPS injections.Conclusions. Our data demonstrate the important role of heparan sulfate and the glycocalyx in endothelial cell activation and suggest a protective role of HPSE2 in microvascular inflammation. HPSE2 offers new options for protection against HPSE1-mediated endothelial damage and preventing microvascular disease.3

Modern integrated photonic platforms should combine low-loss guiding, spectral flexibility, high light confinement, and close packing of optical components. One of the prominent platforms represents a one-dimensional photonic crystal combined with dielectric nanostructures that manipulate low-loss Bloch surface waves (BSWs). Proper design of nanostructures gives rise to a variety of optical resonances suitable for efficient capturing and controlling light. In this work, we achieve color-selective directional excitation of BSWs mediated by Mie resonances in a semiconductor nanoparticle. We show that a single silicon nanoparticle can be used as a subwavelength multiplexer switching the BSW excitation direction from forward to backward within the 30 nm spectral range with its central wavelength governed by the nanoparticle size. Our work opens a route for the on-demand fabrication of photonic nanocouplers with tailored optical properties and submicron footprint.