A methodology for the production of microfabricated electrospun membranes for the creation of new skin regeneration models.

The continual renewal of the epidermis is thought to be related to the presence of populations of epidermal stem cells residing in physically protected microenvironments (rete ridges) directly influenced by the presence of mesenchymal fibroblasts. Current skin in vitro models do acknowledge the influence of stromal fibroblasts in skin reorganisation but the study of the effect of the rete ridge-microenvironment on epidermal renewal still remains a rich topic for exploration. We suggest there is a need for the development of new in vitro models in which to study epithelial stem cell behaviour prior to translating these models into the design of new cell-free biomaterial devices for skin reconstruction. In this study, we aimed to develop new prototype epidermal-like layers containing pseudo-rete ridge structures for studying the effect of topographical cues on epithelial cell behaviour. The models were designed using a range of three-dimensional electrospun microfabricated scaffolds. This was achieved via the utilisation of polyethylene glycol diacrylate to produce a reusable template over which poly(3-hydrroxybutyrate-co-3-hydroxyvalerate) was electrospun. Initial investigations studied the behaviour of keratinocytes cultured on models using plain scaffolds (without the presence of intricate topography) versus keratinocytes cultured on scaffolds containing microfeatures.

Raman spectroscopy detects melanoma and the tissue surrounding melanoma using tissue-engineered melanoma models.

Invasion of melanoma cells from the primary tumor involves interaction with adjacent tissues and extracellular matrix. The extent of this interaction is not fully understood. In this study Raman spectroscopy was applied to cryo-sections of established 3D models of melanoma in human skin. Principal component analysis was used to investigate differences between the tumor and normal tissue and between the peri-tumor area and the normal skin. Two human melanoma cells lines A375SM and C8161 were investigated and compared in 3D melanoma models. Changes were found in protein conformations and tryptophan configurations across the entire melanoma samples, in tyrosine orientation and in more fluid lipid packing only in tumor dense areas, and in increased glycogen content in the peri-tumor areas of melanoma. Raman spectroscopy revealed changes around the perimeter of a melanoma tumor as well as detecting differences between the tumor and the normal tissue.

Arginine functionalization of hydrogels for heparin binding--a supramolecular approach to developing a pro-angiogenic biomaterial.

Our aim was to synthesize a biomaterial that stimulates angiogenesis for tissue engineering applications by exploiting the ability of heparin to bind and release vascular endothelial growth factor (VEGF). The approach adopted involved modification of a hydrogel with positively charged peptides (oligolysine or oligoarginine) to achieve heparin binding. Precursor hydrogels were produced from copolymerization of N-vinyl pyrolidone, diethylene glycol bis allyl carbonate and acrylic acid (PNDA) and functionalized after activation of the carboxylic acid groups with trilysine or triarginine peptides (PNDKKK and PNDRRR). Both hydrogels were shown to bind and release bioactive VEGF165 with arginine-modified hydrogel outperforming the lysine-modified hydrogel. Cytocompatibility of the hydrogels was confirmed in vitro with primary human dermal fibroblasts and human dermal microvascular endothelial cells (HUDMECs). Proliferation of HUDMECs was stimulated by triarginine-functionalized hydrogels, and to a lesser extent by lysine functionalized hydrogels once loaded with heparin and VEGF. The data suggests that heparin-binding hydrogels provide a promising approach to a pro-angiogenic biomaterial.

VEGFR1 receptor tyrosine kinase localization to the Golgi apparatus is calcium-dependent.

Vascular endothelial growth factor receptor 1 (VEGFR1) is an essential receptor tyrosine kinase that regulates mammalian vascular development and embryogenesis but its function is not well understood. Herein, we present evidence whereby endothelial VEGFR1 is largely resident within the Golgi apparatus but translocates to the plasma membrane via a calcium-regulated process. Primary human endothelial cells reveal differing VEGFR1 and VEGFR2 intracellular distribution and dynamics. The major proportion of the full-length VEGFR1 membrane protein was resident within the Golgi apparatus in primary endothelial cells. Whereas VEGFR2 displayed down-regulation in response to VEGF-A, VEGFR1 was not significantly affected arguing for a significant intracellular pool that was inaccessible to extracellular VEGF-A. This intracellular VEGFR1 pool showed significant co-distribution with key Golgi residents. Brefeldin A caused VEGFR1 Golgi fragmentation consistent with redistribution to the endoplasmic reticulum. Metabolic labeling experiments and microscopy using domain-specific VEGFR1 antibodies indicated that the mature processed VEGFR1 species and an integral membrane protein was resident within Golgi apparatus. Cytosolic calcium ions play a key role in VEGFR1 trafficking as treatment with either VEGF-A, histamine, thrombin, thapsigargin or A23187 ionophore caused VEGFR1 redistribution from the Golgi apparatus to small punctate vesicles and plasma membrane. We thus propose a model whereby the balance of VEGFR1 and VEGFR2 plasma membrane levels dictate either negative or positive endothelial signaling to influence vascular physiology.

Intrinsic tyrosine kinase activity is required for vascular endothelial growth factor receptor 2 ubiquitination, sorting and degradation in endothelial cells.

The human endothelial vascular endothelial growth factor receptor 2 (VEGFR2/kinase domain region, KDR/fetal liver kinase-1, Flk-1) tyrosine kinase receptor is essential for VEGF-mediated physiological responses including endothelial cell proliferation, migration and survival. How VEGFR2 kinase activation and trafficking are co-coordinated in response to VEGF-A is not known. Here, we elucidate a mechanism for endothelial VEGFR2 response to VEGF-A dependent on constitutive endocytosis co-ordinated with ligand-activated ubiquitination and proteolysis. The selective VEGFR kinase inhibitor, SU5416, blocked the endosomal sorting required for VEGFR2 trafficking and degradation. Inhibition of VEGFR2 tyrosine kinase activity did not block plasma membrane internalization but led to endosomal accumulation. Lysosomal protease activity was required for ligand-stimulated VEGFR2 degradation. Activated VEGFR2 codistributed with the endosomal hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)/signal-transducing adaptor molecule (STAM) complex in a ligand and time-dependent manner, implying a role for this factor in sorting of ubiquitinated VEGFR2. Increased tyrosine phosphorylation of the Hrs subunit in response to VEGF-A links VEGFR2 activation and Hrs/STAM function. In contrast, VEGFR2 in quiescent cells was present on both the endothelial plasma membrane and early endosomes, suggesting constitutive recycling between these two compartments. This pathway was clathrin-linked and dependent on the AP2 adaptor complex as the A23 tyrphostin inhibited VEGFR2 trafficking. We propose a mechanism whereby the transition of endothelial VEGFR2 from a constitutive recycling itinerary to a degradative pathway explains ligand-activated receptor degradation in endothelial cells. This study outlines a mechanism to control the VEGF-A-mediated response within the vascular system.

Endothelial cell confluence regulates Weibel-Palade body formation.

Secretory granules called Weibel-Palade bodies (WPBs) containing Von Willebrand factor (VWF) are characteristic of the mammalian endothelium. We hypothesized that vascular-specific antigens such as VWF are linked to endothelial identity and proliferation in vitro. To test this idea, the cellular accumulation of VWF in WPBs was monitored as a function of cell proliferation, confluence and passage number in human umbilical vein endothelial cells (HUVECs). We found that as passage number increased the percentage of cells containing VWF in WPBs was reduced significantly, whilst the protein was still detected within the secretory pathway at all times. However, the endothelial-specific marker protein, PECAM-1, is present on all cells even when WPBs are absent, indicating partial maintenance of endothelial identity. Biochemical studies show that a significant pool of immature pro-VWF can be detected in sub-confluent HUVECs; however, a larger pool of mature, processed VWF is detected in confluent cells. Newly synthesized VWF must thus be differentially sorted and packaged along the secretory pathway in semi-confluent versus confluent endothelial cells. Our studies thus show that WPB formation is linked to the formation of a confluent endothelial monolayer.