Platelet degranulation and bleeding phenotype in a large cohort of Von Willebrand disease patients

Von Willebrand disease (VWD) is a bleeding disorder caused by quantitative (type 1 or 3) or qualitative (type 2A/2B/2M/2N) defects of circulating von Willebrand factor (VWF). Circulating VWF levels not always fully explain bleeding phenotypes, suggesting a role for alternative factors, like platelets. Here, we investigated platelet factor 4 (PF4) in a large cohort of patients with VWD. PF4 levels were lower in type 2B and current bleeding phenotype was significantly associated with higher PF4 levels, particularly in type 1 VWD. Based on our findings we speculate that platelet degranulation and cargo release may play a role across VWD subtypes

GDP/GTP exchange factor MADD drives activation and recruitment of secretory Rab GTPases to Weibel-Palade bodies

von Willebrand factor (VWF) is an essential hemostatic protein that is synthesized and secreted by endothelial cells and stored in Weibel-Palade bodies (WPBs). The secretory Rab GTPases Rab27A, Rab3B, and Rab3D have been linked with WPB trafficking and secretion. How these Rabs are activated and recruited to WPBs remains elusive. In this study, we identified MAP kinase-activating death domain (MADD) as the guanine nucleotide exchange factor for Rab27A and both Rab3 isoforms in primary human endothelial cells. Rab activity assays revealed a reduction in Rab27A, Rab3B, and Rab3D activation upon MADD silencing. Rab activation, but not binding, was dependent on the differentially expressed in normal and neoplastic cells (DENN) domain of MADD, indicating the potential existence of 2 Rab interaction modules. Furthermore, immunofluorescent analysis showed that Rab27A, Rab3B, and Rab3D recruitment to WPBs was dramatically decreased upon MADD knockdown, revealing that MADD drives Rab membrane targeting. Artificial mistargeting of MADD using a TOMM70 tag abolished Rab27A localization to WPB membranes in a DENN domain-dependent manner, indicating that normal MADD localization in the cytosol is crucial. Activation of Rab3B and Rab3D was reduced upon Rab27A silencing, suggesting that activation of these Rabs is enhanced through previous activation of Rab27A by MADD. MADD silencing did not affect WPB morphology, but it did reduce VWF intracellular content. Furthermore, MADD-depleted cells exhibited decreased histamine-evoked VWF release, similar to Rab27A-depleted cells. In conclusion, MADD acts as a master regulator of VWF secretion by coordinating the activation and membrane targeting of secretory Rabs to WPBs

A Printable Photopolymerizable Thermosensitive p(HPMAm-lactate)-PEG Hydrogel for Tissue Engineering

Bioprinting is a new technology in regenerative medicine that allows the engineering of tissues by specific placement of cells in biomaterials. Importantly, the porosity and the relatively small dimensions of the fibers allow rapid diffusion of nutrients and metabolites. This technology requires the availability of hydrogels that ensure viability of encapsulated cells and have adequate mechanical properties for the preparation of structurally stable and well-defined three-dimensional constructs. The aim of this study is to evaluate the suitability of a biodegradable, photopolymerizable and thermosensitive A–B–A triblock copolymer hydrogel as a synthetic extracellular matrix for engineering tissues by means of three dimensional fiber deposition. The polymer is composed of poly(N-(2-hydroxypropyl)methacrylamide lactate) A-blocks, partly derivatized with methacrylate groups, and hydrophilic poly(ethylene glycol) B-blocks of a molecular weight of 10 kDa. Gels are obtained by thermal gelation and stabilized with additional chemical cross-links by photopolymerization of the methacrylate groups coupled to the polymer. A power law dependence of the storage plateau modulus of the studied hydrogels on polymer concentration is observed for both thermally and chemically cross-linked hydrogels. The hydrogels demonstrated mechanical characteristics similar to natural semi-flexible polymers, including collagen. Moreover, the hydrogel shows suitable mechanical properties for bioprinting, allowing subsequent layer-by-layer deposition of gel fibers to form stable constructs up to at least 0.6 cm (height) with different patterns and strand spacing. The resulting constructs have reproducible vertical porosity and the ability to maintain separate localization of encapsulated fluorescent microspheres. Moreover, the constructs show an elastic modulus of 119 kPa (25 wt% polymer content) and a degradation time of approximately 190 days. Furthermore, high viability is observed for encapsulated chondrocytes after 1 and 3 days of culture. In summary, we conclude that the evaluated hydrogel is an interesting candidate for bioprinting applications

The complex mural cell : Pericyte function in health and disease

Pericytes are perivascular cells that can be distinguished from vascular smooth muscle cells by their specific morphology and expression of distinct molecular markers. Found in the microvascular beds distributed throughout the body, they are well known for their regulation of a healthy vasculature. In this review, we examine the mechanism of pericyte support to vasomotion, and the known pathways that regulate pericyte response in angiogenesis and neovascular stabilization. We will also discuss the role of pericytes in vascular basement membrane and endothelial barrier function regulation. In contrast, recent findings have indicated that pericyte dysfunction, characterized by changes in pericyte contractility or pericyte loss of microvascular coverage, plays an important role in onset and progression of vascular-related and fibrogenic diseases. From a therapeutic point of view, pericytes have recently been identified as a putative pool of endogenous mesenchymal stem cells that could be activated in response to tissue injury to contribute to the regenerative process on multiple levels. Wewill discuss the mechanisms viawhich pericytes are involved in disease onset and development in a number of pathophysiological conditions, as well as present the evidence that supports a role for multipotent pericytes in tissue regeneration. The emerging field of pericyte research will not only contribute to the identification of new drug targets in pericyte dysfunction associated diseases, but may also boost the use of this cell type in future cell-based regenerative strategies